A novel cellobiose 2-epimerase from anaerobic halophilic Iocasia fonsfrigidae and its ability to convert lactose in fresh goat milk into epilactose.

BACKGROUND: Cellobiose 2-epimerase (CE) has received great attention due to its potential applications in the food and pharmaceutical industries. In this study, a novel CE from mesophilic anaerobic halophilic bacterium Iocasia fonsfrigidae strain SP3-1 (IfCE) was successfully expressed in Escherichia coli and characterized. RESULTS: Unlike other CEs, the purified IfCE shows only epimerization activity toward ß-1,4-glycosidic linkages of disaccharides, including mannobiose, cellobiose and lactose, but not for monosaccharides, ß-1,4-glycosidic linkages of trisaccharides and α-1,4-glycosidic linkages of disaccharides. Only one epimerization product was obtained from the action of IfCE against mannobiose, cellobiose and lactose. Under optimum conditions, 31.0% of epilactose, a rare and low-calorie prebiotic sweetener with medicinal and pharmacological properties, was obtained from 10 mg mL-1 lactose. IfCE was highly active against lactose under NaCl concentrations up to 500 mmol L-1, possibly due to the excessive basic (arginine and lysine) and acidic (aspartic and glutamic acids) amino acid residues, which are localized on the surface of the halophilic enzyme structure. These residues may protect the enzyme from Cl- and Na+ ions from the environment, respectively. Under normal conditions, IfCE was able to convert lactose present in fresh goat milk to epilactose with a conversion yield of 31% in 10 min. In addition, IfCE has been investigated as a safe enzyme for human allergen. CONCLUSION: The results suggested that IfCE is a promising candidate to increase the quality and value of milk and dairy products by converting lactose that causes digestive problems in people with lactose intolerance into epilactose. © 2024 Society of Chemical Industry.

Investigations on the Impacts of Drugs or Excipients with Different Physicochemical and Compaction Properties on the Disintegration Behavior of Kollidon®SR-Based Binary Controlled Release Matrix Tablets.

The objective of this study was to examine the impact of the physicochemical properties of the loaded drug or excipient, the concentration of Kollidon®SR (KSR), and the mechanical characteristics of KSR compacts on their disintegration times. Using disintegration apparatus, a two-hour constraint was chosen as the process's end point. Lactose-KSR compacts subjected to the highest compression pressure and Microcrystalline cellulose-KSR compacts with KSR concentrations exceeding 30% exhibited disintegration times of less than ten minutes. Likewise, compacts containing Diltiazem HCl-KSR demonstrated brief disintegration times across all tested KSR concentrations and compression pressures. Compacts of Modafinil, Metformin HCl, and Ascorbic acid-KSR displayed disintegration times ranging from fast to moderate, contingent upon the levels of KSR and compression pressure applied. Compacts containing KSR with Aspirin, Salicylic acid, or Ibuprofen did not exhibit significant disintegration even at minimal amounts of KSR (0.5%). Theophylline-KSR tablets also showed prolonged dissolution times, even at very low concentrations of KSR. The disintegration times of Dic-KSR tablets were roughly close to an hour and were predominantly unaffected by varying KSR levels and only marginally influenced by compression pressures. It is possible to draw the conclusion that different drugs or excipients have different minimum KSR requirements to resist compacts' disintegration process. Compounds that demonstrate low solubility in water can result in extended disintegration times for KSR compacts. The melting points of these compounds, in conjunction with the Py values of the compacts and their compaction properties, could affect the disintegration process, although a precise evaluation is necessary.

Recombinant production of Paenibacillus wynnii ß-galactosidase with Komagataella phaffii.

BACKGROUND: The ß-galactosidase from Paenibacillus wynnii (ß-gal-Pw) is a promising candidate for lactose hydrolysis in milk and dairy products, as it has a higher affinity for the substrate lactose (low KM value) compared to industrially used ß-galactosidases and is not inhibited by the hydrolysis-generated product D-galactose. However, ß-gal-Pw must firstly be produced cost-effectively for any potential industrial application. Accordingly, the yeast Komagataella phaffii was chosen to investigate its feasibility to recombinantly produce ß-gal-Pw since it is approved for the regulated production of food enzymes. The aim of this study was to find the most suitable way to produce the ß-gal-Pw in K. phaffii either extracellularly or intracellularly. RESULTS: Firstly, 11 different signal peptides were tested for extracellular production of ß-gal-Pw by K. phaffii under the control of the constitutive GAP promoter. None of the signal peptides resulted in a secretion of ß-gal-Pw, indicating problems within the secretory pathway of this enzyme. Therefore, intracellular ß-gal-Pw production was investigated using the GAP or methanol-inducible AOX1 promoter. A four-fold higher volumetric ß-galactosidase activity of 7537 ± 66 µkatoNPGal/Lculture was achieved by the K. phaffii clone 27 using the AOX1 promoter in fed-batch bioreactor cultivations, compared to the clone 5 using the GAP promoter. However, a two-fold higher specific productivity of 3.14 ± 0.05 µkatoNPGal/gDCW/h was achieved when using the GAP promoter for ß-gal-Pw production compared to the AOX1 promoter. After partial purification, a ß-gal-Pw enzyme preparation with a total ß-galactosidase activity of 3082 ± 98 µkatoNPGal was obtained from 1 L of recombinant K. phaffii culture (using AOX1 promoter). CONCLUSION: This study showed that the ß-gal-Pw was produced intracellularly by K. phaffii, but the secretion was not achieved with the signal peptides chosen. Nevertheless, a straightforward approach to improve the intracellular ß-gal-Pw production with K. phaffii by using either the GAP or AOX1 promoter in bioreactor cultivations was demonstrated, offering insights into alternative production methods for this enzyme.

An efficient approach for overproduction of DNA polymerase from Pyrococcus furiosus using an optimized autoinduction system in Escherichia coli.

High fidelity DNA polymerase from Pyrococcus furiosus (Pfupol) is an attractive alternative to the highly popular DNA polymerase from Thermus aquaticus. Because this enzyme is in great demand for biotechnological applications, optimizing Pfupol production is essential to supplying the industry's expanding demand. T7-induced promoter expression in Escherichia coli expression systems is used to express recombinant Pfupol; however, this method is not cost-effective. Here, we have effectively developed an optimized process for the autoinduction approach of Pfupol expression in a defined medium. To better examine Pfupol's activities, its purified fraction was used. A 71 mg/L of pure Pfupol was effectively produced, resulting in a 2.6-fold increase in protein yield when glucose, glycerol, and lactose were added in a defined medium at concentrations of 0.05%, 1%, and 0.6%, respectively, and the condition for production in a 5 L bioreactor was as follow: 200 rpm, 3 vvm, and 10% inoculant. Furthermore, the protein exhibited 1445 U/mg of specific activity when synthesized in its active state. This work presents a high level of Pfupol production, which makes it an economically viable and practically useful approach.

Chemiosmotic nutrient transport in synthetic cells powered by electrogenic antiport coupled to decarboxylation.

Cellular homeostasis depends on the supply of metabolic energy in the form of ATP and electrochemical ion gradients. The construction of synthetic cells requires a constant supply of energy to drive membrane transport and metabolism. Here, we provide synthetic cells with long-lasting metabolic energy in the form of an electrochemical proton gradient. Leveraging the L-malate decarboxylation pathway we generate a stable proton gradient and electrical potential in lipid vesicles by electrogenic L-malate/L-lactate exchange coupled to L-malate decarboxylation. By co-reconstitution with the transporters GltP and LacY, the synthetic cells maintain accumulation of L-glutamate and lactose over periods of hours, mimicking nutrient feeding in living cells. We couple the accumulation of lactose to a metabolic network for the generation of intermediates of the glycolytic and pentose phosphate pathways. This study underscores the potential of harnessing a proton motive force via a simple metabolic network, paving the way for the development of more complex synthetic systems.

Untargeted metabolomic analysis of human milk from healthy mothers reveals drivers of metabolite variability.

Understanding the human milk metabolome can help inform infant nutrition and health. Untargeted metabolomics was used to study breast milk from 31 healthy participants to assess the shared metabolites in milk from participants with various backgrounds and understand how different demographic, health, and environmental factors impact the milk metabolome. Breast milk samples were analyzed by four separate UPLC-MS/MS methods. Metabolite Set Enrichment Analysis was used to study the most and least variable metabolites. The associations between participant factors and the metabolome were assessed with redundancy analyses. Among all 31 participants and between each untargeted UPLC-MS/MS method, 731 metabolites were detected, of which 389 were shared among all participants. Of the shared metabolites, lactose was the least and lactobionate the most variable metabolite. In the biological super pathway analysis, xenobiotics were the most variable metabolites. Infant age, maternal age, number of live births, and pre-pregnancy BMI were associated with the milk metabolome. In conclusion, the most variable metabolites originate from environmental exposures while the well-conserved core metabolites are linked to cell metabolism or are crucial for infant nutrition and osmoregulation. Understanding the variability of the breast milk metabolome can help identify components that are crucial for infant nutrition, growth, and development.

Rational modification of Neisseria meningitidis ß1,3-N-acetylglucosaminyltransferase for lacto-N-neotetraose synthesis with reduced long-chain derivatives.

Lacto-N-neotetraose (LNnT), as a neutral core structure within human milk oligosaccharides (HMOs), has garnered widespread attention due to its exceptional physiological functions. In the process of LNnT synthesis using cellular factory approaches, substrate promiscuity of glycosyltransferases leads to the production of longer oligosaccharide derivatives. Here, rational modification of ß1,3-N-acetylglucosaminyltransferase from Neisseria meningitidis (LgtA) effectively decreased the concentration of long-chain LNnT derivatives. Specifically, the optimal ß1,4-galactosyltransferase (ß1,4-GalT) was selected from seven known candidates, enabling the efficient synthesis of LNnT in Escherichia coli BL21(DE3). Furthermore, the influence of lactose concentration on the distribution patterns of LNnT and its longer derivatives was investigated. The modification of LgtA was conducted with computational assistance, involving alanine scanning based on molecular docking to identify the substrate binding pocket and implementing large steric hindrance on crucial amino acids to obstruct LNnT entry. The implementation of saturation mutagenesis at positions 223 and 228 of LgtA yielded advantageous mutant variants that did not affect LNnT synthesis while significantly reducing the production of longer oligosaccharide derivatives. The most effective mutant, N223I, reduced the molar ratio of long derivatives by nearly 70 %, showcasing promising prospects for LNnT production with diminished byproducts.

Human milk oligosaccharides are associated with maternal genetics and respiratory health of human milk-fed children.

Breastfeeding provides many health benefits, but its impact on respiratory health remains unclear. This study addresses the complex and dynamic nature of the mother-milk-infant triad by investigating maternal genomic factors regulating human milk oligosaccharides (HMOs), and their associations with respiratory health among human milk-fed infants. Nineteen HMOs are quantified from 980 mothers of the CHILD Cohort Study. Genome-wide association studies identify HMO-associated loci on chromosome 19p13.3 and 19q13.33 (lowest P = 2.4e-118), spanning several fucosyltransferase (FUT) genes. We identify novel associations on chromosome 3q27.3 for 6'-sialyllactose (P = 2.2e-9) in the sialyltransferase (ST6GAL1) gene. These, plus additional associations on chromosomes 7q21.32, 7q31.32 and 13q33.3, are replicated in the independent INSPIRE Cohort. Moreover, gene-environment interaction analyses suggest that fucosylated HMOs may modulate overall risk of recurrent wheeze among preschoolers with variable genetic risk scores (P < 0.01). Thus, we report novel genetic factors associated with HMOs, some of which may protect the respiratory health of children.

Bovine Highly Pathogenic Avian Influenza Virus Stability and Inactivation in the Milk Byproduct Lactose.

The recent incursion of highly pathogenic influenza viruses into dairy cattle opens new insights for influenza virus ecology and its interspecies transmission and may have a significant impact on public health and agriculture. The aim of this study was to determine the stability of a bovine highly pathogenic avian influenza H5N1 virus isolate in the milk byproduct lactose and to evaluate two inactivation methods using industrial procedures. The bovine isolate of the highly pathogenic avian influenza H5N1 virus was stable for 14 days in a concentrated lactose solution under refrigerated conditions. Heat or citric acid treatments successfully inactivated the virus in lactose. This study highlights the persistence of HPAIV in lactose and its efficient inactivation under industrial standards.

Experimental investigation of dynamic drying in single pharmaceutical granules containing acetaminophen or carbamazepine using synchrotron X-ray micro computed tomography.

Drying time, velocity, and temperature are important aspects of the drying process for pharmaceutical granules observed during tablet manufacturing. However, the drying mechanism of single granules is often limited to modelling and simulation, with the internal and physical changes difficult to quantify at an experimental level. In this study, in-situ synchrotron-based X-ray imaging techniques were used for the first time to investigate the dynamic drying of single pharmaceutical granules, quantifying internal changes occurring over the drying time. Two commonly used excipients (lactose monohydrate (LMH) and microcrystalline cellulose (MCC)) were used as pure components and binary mixtures with one of either two active pharmaceutical ingredients of differing hydrophilicity/hydrophobicity (acetaminophen (APAP) and carbamazepine (CBZ)). Water was used as a liquid binder to generate single granules of 25 % to 30 % moisture content. Results showed that for most samples, the drying time and composition significantly influences the pore volume evolution and the moisture ratio, with the velocity and temperature of the drying air possessing mixed significance on increasing the rate of pore connectivity and moisture removal depending on the sample composition. Effects of active ingredient loading resulted in minimal influence on the drying of CBZ and generated binary mixtures, with APAP and its respective mixtures' drying behaviour dominated by the material's hydrophilic nature.

6'-Sialyllactose Alleviates Muscle Fatigue through Reduced Blood Lactate Level after Treadmill Exercise in Mice.

6'-Sialyllactose (6'-SL), found in human breast milk, exhibits anti-inflammatory, immune function-enhancing, brain development-promoting, and gut health-improving effects. However, its effects on muscle fatigue remain unknown. Here, we aimed to investigate the effects of 6'-SL on blood lactate level, muscle fiber type, and oxidative phosphorylation protein complexes (OXPHOS) in muscle after exercise using C57BL/6J male mice. C57BL/6J mice were randomly assigned to control or 100 mg/kg 6'-SL. After 12 weeks of 6'-SL administration, the mice were made to perform treadmill exercise; their blood lactate and glucose levels were measured at the basal level (rest) and 0, 5, and 10 min after treadmill exercise. Results showed that 6'-SL treatment in C57BL/6J mice significantly reduced blood lactate level and improved blood glucose level. Moreover, 6'-SL increased the expression of slow-myosin heavy chain (MHC) and OXPHOS in gastrocnemius muscle. In addition, 6'-SL treatment for 12 weeks did not affect food intake, serum biomarkers of tissue injury, and lipid profiles compared with those of the controls. These findings indicate that non-toxic 6'-SL suppressed muscle fatigue during exercise by promoting protein expression of muscle fibers, especially slow-twitch muscle fibers characterized by abundant OXPHOS complexes and decreased blood lactate level. This study suggests that 6'-SL holds promise as a nutritional supplement in exercise and clinical settings, subject to further validation.

[Research progress in the synthesis of 3'- and 6'-sialactose by Escherichia coli].

Human milk oligosaccharides (HMOs) are a structurally complex group of unbound polysaccharides, representing the third-largest solid component in breast milk. They play a crucial role in the intestinal health and immune system development of infants. Sialylated HMOs, including 3'-sialactose (3'-SL) and 6'-sialactose (6'-SL), are major components of HMOs, playing significant roles in immune regulation, anti-inflammatory processes, and promotion of probiotic growth. Currently, the cost-effective production of high-value sialactose by microbial fermentation with readily available raw materials has become a research hotspot due to the high nutritional value and potential applications of sialylated HMOs in infant food. This paper summarizes the functions and biosynthesis of 3'-SL and 6'-SL. Furthermore, it reviews the research progress in the synthesis of sialactose by Escherichia coli, offering valuable insights for future industrial production.

Biopharmaceutical studies of a novel sedative sublingual lozenge based on glycine and tryptophan: A rationale for mucoadhesive agent selection.

Effective sedative drugs are in great demand due to increasing incidence of nervous disorders. The present work was aimed to develop a novel sublingual sedative drug based on glycine and L-tryptophan amino acids. Carbopol and different hydroxypropyl methylcellulose species were alternatively tested as mucoadhesive agents intended to prolong tryptophan sublingual release time. A model lipid medium of fully hydrated L-α-dimyristoylphosphatidylcholine was used for optimal mucoadhesive agents selection. Simultaneous processes of drug release and diffusion in lipid medium were first investigated involving both experimental and theoretical approaches. Individual substances, their selected combinations as well as different drug formulations were consecutively examined. Application of kinetic differential scanning calorimetry method allowed us to reveal a number of specific drug-excipient effects. Lactose was found to essentially facilitate tryptophan release and provide its ability to get into the bloodstream simultaneously with glycine, which is necessary to achieve glycine-tryptophan synergism. Introduction of a mucoadhesive agent into the formulation was shown to change kinetics of drug-membrane interactions variously depending on viscosity grade. Among the mucoadhesive agents, hydroxypropyl methylcellulose species K4M and E4M were shown to further accelerate drug release, therefore they were selected as optimal. Thus, effectiveness of the novel sedative drug was provided by including some excipients, such as lactose and the selected mucoadhesive agent species. A dynamic mathematical model was developed properly describing release and diffusion in lipid medium of various drug substances. Our study clearly showed applicability of a lipid medium to meet challenges such as drug-excipient interactions and optimization of drug formulations.

Optimizing dsRNA engineering strategies and production in E. coli HT115 (DE3).

Producing double-stranded RNA (dsRNA) represents a bottleneck for the adoption of RNA interference technology in agriculture, and the main hurdles are related to increases in dsRNA yield, production efficiency, and purity. Therefore, this study aimed to optimize dsRNA production in E. coli HT115 (DE3) using an in vivo system. To this end, we designed a new vector, pCloneVR_2, which resulted in the efficient production of dsRNA in E. coli HT115 (DE3). We performed optimizations in the culture medium and expression inducer in the fermentation of E. coli HT115 (DE3) for the production of dsRNA. Notably, the variable that had the greatest effect on dsRNA yield was cultivation in TB medium, which resulted in a 118% increase in yield. Furthermore, lactose induction (6 g/L) yielded 10 times more than IPTG. Additionally, our optimized up-scaled protocol of the TRIzol™ extraction method was efficient for obtaining high-quality and pure dsRNA. Finally, our optimized protocol achieved an average yield of 53.3 µg/mL after the production and purification of different dsRNAs, reducing production costs by 72%.

Computer-aided mining of a psychrophilic cellobiose 2-epimerase from the Qinghai-Tibet Plateau gene catalogue.

Cellobiose 2-epimerase (CE) catalyzes the conversion of the lactose into its high-value derivatives, epilactose and lactulose, which has great prospects in food applications. In this study, CE sequences from the Qinghai-Tibet Plateau gene catalogue, we screened these for structural flexibility through molecular dynamics simulation to identify potential psychrophilic CE candidates. One such psychrophilic CE we termed psyCE demonstrated exceptional epimerization activity, achieving an optimum activity of 122.2 ± 1.6 U/mg. Its kinetic parameters (Kcat and Km) for epimerization activity were 219.9 ± 5.6 s-1 and 261.9 ± 18.1 mM, respectively, representing the highest Kcat recorded among known cold-active CEs. Notably, this is the first report of a psychrophilic CE. The psyCE can effectively produce epilactose at 8 °C, converting 20.3 % of 200 mM lactose into epilactose within four hours. These findings suggest that psyCE is highly suitable for cryogenic food processing, and glaciers may serve as a valuable repository of psychrophilic enzymes.

New theory to explain the effect of lactose fines on the performance of adhesive mixtures for inhalation.

A new theory for the dispersibility enhancing effect of excipient fines for adhesive mixtures for inhalation is presented in this paper, while at the same time the shortcomings of current hypotheses are discussed. The proposed mechanism, denoted the 'viscoelastic damping effect', states that the presence of fines particles acts to dampen the collisions between carrier particles during mixing. As a consequence, fewer fine particles are 'irreversibly' pressed into the carriers, which in turn entails a higher fine particle fraction. The mechanism was demonstrated experimentally at different levels of added lactose fines by studying the influence of processing on fine particle fraction. This approach furthermore enabled quantification of the effect. All fine particles present in the blend (APIs and excipient fines) act together to exert the damping effect. The proposed mechanism is able to explain the main body of published data, including the effect of added excipient fines, the effect of an increased drug load, and the effect of removal of carrier fines. The viscoelastic damping mechanism is general in nature and conveys a broader and more general understanding of the behavior of adhesive mixtures for inhalation.

An interaction-based mixing model for predicting porosity and tensile strength of directly compressed ternary blends of pharmaceutical powders.

Predicting the mechanical properties of powder mixtures without extensive experimentation is important for model driven design in solid dosage form manufacture. Here, a new binary interaction-based model is proposed for predicting the compressibility and compactability of directly compressed pharmaceutical powder mixtures based on the mixture composition. The model is validated using blends of MCC, lactose and paracetamol or ibuprofen. Both compressibility and compactability profiles are predicted well for a variety of blend compositions of ternary mixtures for the two formulations. The model performs well over a wide range of compositions for both blends and better than either an ideal mixing model or a ternary interaction model. A design of experiments which reduces the amount of API required for fitting the model parameters for a new formulation is proposed to reduce amount of API required. The design requires only three blends containing API. The model gives similar performance to the well-known Reynolds et al. model (2017) when trained using the same data sets. The binary interaction model approach is generalizable to other powder mixture properties. The model presented in this work is limited to curve-fitting of empirical compaction models for mixtures of common pharmaceutical powders and is not intended to provide guidance on the practical operating space (or design space) limits.

Synergistic effect of magnesium stearate and fine lactose in improving aerosolization performance of fluticasone propionate in dry powder formulation.

Magnesium stearate (MgSt) and lactose fines are often used as ternary components in carrier-based dry powder inhalers (DPIs) to improve fine particle fraction (FPF), but whether they act synergistically to improve aerosolization performance of DPI formulations is currently less studied. In addition, the applicability of utilizing powder rheological parameters to predict the FPF needs to be further verified. Thus, in this study, using fluticasone propionate (FP) as a model drug, effect of lactose fines addition in 0.5% MgSt containing DPI formulations on their powder and aerodynamic properties was explored. Influence of MgSt and fines mixing order on the DPIs performance was also investigated. The results showed that addition of lactose fines (1-10%) in 0.5% MgSt containing formulations could further improve flowability and enhance adhesion of the mixtures, and they could act synergistically to improve FPF. Moreover, the presence of 0.5% MgSt can greatly reduce the amount of lactose fines required to achieve the comparable FPF. The mixing order can affect distribution of MgSt on the carrier surface, with higher FPF noted when MgSt was mixed with carrier first, followed by lactose fines. A good linear relationship between powder rheological parameters such as basic flowability energy (BFE), Permeability and FPF was disclosed. In conclusion, in FP based DPIs, MgSt and lactose fines act synergistically to enhance FPF by tuning powder characteristics. Good flowability (27.39%) and strong adhesion (72.61%) contributed to the enhanced drug deposition in the lung.

Enhancement of ß-galactosidase catalytic activity and stability through covalent immobilization onto alginate/tea waste beads and evaluating its impact on the quality of some dairy products.

The current study aimed to evaluate the hydrolysis of whole fat milk (WFM) and sweet whey (SW) using ß-galactosidase (ß-gal) after covalent immobilization onto activated alginate/tea waste (Alg/TW) beads as a novel carrier. The optimum temperature for free and Alg/TW/ß-gal was 40 °C and the ideal pH was 7.0. However, Alg/TW/ß-gal displayed better stabilities at high temperatures and a wide pH range. Additionally, the value of Km and Vmax for Alg/TW/ß-gal was higher than the free enzyme. The Alg/TW/ß-gal showed better residual activity (78.6 %) after 90 storage days at 4 °C. The reusability of Alg/TW/ß-gal was very good as it conserved its full activity after 15 consecutive cycles and conserved 93 % of its initial activity after 10 cycles with ONPG (O-nitrophenyl-ß-D-galactopyranoside) and lactose as a substrate, respectively. The impact of Alg/TW/ß-gal on WFM and SW using HPLC analysis revealed a remarkable decrease in lactose concentration and increase of glucose and galactose concentrations. The SW exhibited higher degree of lactose hydrolysis (97.3 %) compared to WFM (62.4 %). Besides, SW had a prominent increase in total phenolic content (96.8 mg/L) compared to WFM (54.3 mg/L). The antioxidant activity had increased after enzyme treatment in both WFM and SW. The GC-MS analysis for volatile compounds identified twenty-five flavour constituents. Finally, Alg/TW/ß-gal has a potential application for obtaining healthy, acceptable, and commercial dairy products of low lactose.

Non-canonical start codons confer context-dependent advantages in carbohydrate utilization for commensal E. coli in the murine gut.

Resource competition is a driver of gut microbiota composition. Bacteria can outcompete metabolically similar rivals through the limitation of shared growth-fuelling nutrients. The mechanisms underlying this remain unclear for bacteria with identical sets of metabolic genes. Here we analysed the lactose utilization operon in the murine commensal Escherichia coli 8178. Using in vitro and in vivo approaches, we showed that translation of the lactose utilization repressor gene lacI from its native non-canonical GTG start codon increases the basal expression of the lactose utilization cluster, enhancing adaptation to lactose consumption. Consequently, a strain carrying the wild type lacI GTG start codon outperformed the lacI ATG start codon mutant in the mouse intestine. This advantage was attenuated upon limiting host lactose intake through diet shift or altering the mutant frequency, emphasizing the context-dependent effect of a single nucleotide change on the bacterial fitness of a common member of the gut microbiota. Coupled with a genomic analysis highlighting the selection of non-ATG start codons in sugar utilization regulator genes across the Enterobacteriaceae family, our data exposed an unsuspected function of non-canonical start codons in metabolic competition.