Thiamin biofortification of food crops by genetic engineering has the potential to solve human health issues related to thiamin deficiencies. Rice is an obvious target for thiamin biofortification because white rice, which is obtained after removing the thiamin-rich outer layer of the grain, is a poor source of thiamin. To increase the levels and stability of thiamin in white rice, engineering strategies that boost the expression of biosynthesis genes and/or thiamin binding proteins have been reported. In this chapter, we describe protocols for generating and characterizing thiamin-engineered plants: (1) design and building of plant expression vectors, (2) introduction of DNA constructs into plant genome by Agrobacterium-mediated transformation, (3) analysis of transgene expression by quantitative reverse-transcription polymerase chain reaction, (4) quantification of thiamin biosynthesis proteins by Western blot, and (5) quantification of thiamin by HPLC.

This chapter describes a single-molecule fluorescence resonance energy transfer (smFRET) approach to monitor cotranscriptional RNA folding within native transcription complexes. The approach combines stepwise transcription by the Escherichia coli RNA polymerase (RNAP) with site-specific RNA labeling with a Cy3-Cy5 FRET pair via copper-free click chemistry. The chapter also details single molecule approaches to monitor the structural aspects of nascent transcripts and the analysis of observed RNA populations. Using the thiamin pyrophosphate (TPP)-sensing E. coli tbpA riboswitch as a model system, we detail procedures for DNA template preparation, RNA labeling and smFRET acquisition. Key advantages of the described smFRET approach include the use of native transcripts and the ability to monitor RNA structural changes within transcription complexes that are either stalled at naturally occurring pause sites or that are actively elongating along the DNA template. This method permits the study of cotranscriptional regulatory mechanisms taking place either in bacterial or eukaryotic elongation complexes and could be extrapolated to the use of any biochemical probes compatible with the click chemistry approach.

This chapter describes the use of transcriptome-wide sequencing to identify ligand-RNA interactions. Photoaffinity probes, based on small molecules of interest, can directly report on binding of ligands to their target RNA. Combined with pulldown enrichment experiments and next-generation sequencing (NGS), this chemical tool allows for transcriptome-wide screening to identify novel RNA targets. Here, we describe protocols to identify and validate ligand-RNA interactions using pulldown enrichments followed by transcriptome-wide sequencing or Reverse Transcription-quantitative PCR (RT-qPCR), and to investigate these interactions using Reverse-Transcription (RT) stop, Selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE) and in vitro transcription and translation (IVTT) assays.

Thiamine (vitamin B1) biosynthesis involves enzymatic activities that exhibit remarkable diversity across the tree of life, including deep sequence divergence, domain rearrangements, lineage-specific duplications and losses, and horizontal gene transfer events. These features complicate computational inference based on single evidence types. This chapter describes a reproducible, modular bioinformatics workflow for pathway-scale comparative analysis of thiamine biosynthesis genes across Bacteria, Archaea, and Eukarya. The workflow integrates four complementary evidence streams: (i) profile-based homolog discovery using Hidden Markov Models (HMMs) with domain architecture validation; (ii) per-family phylogenetic reconstruction to evaluate evolutionary relationships, distinguish orthologs from paralogs, and identify lineage-specific patterns; (iii) detection of TPP riboswitches using covariance models, with an overview of upstream promoter motif discovery as a complementary observational approach; and (iv) transcriptome integration to assess context-dependent gene expression. We further demonstrate how variation in protein domain architecture, particularly single versus multi domain configurations, shape sequence recovery, phylogenetic inference, and evolutionary interpretation. Step-by-step protocols cover computational environment setup, parameter optimization, quality control checkpoints, and interpretation guidelines. The workflow addresses common bioinformatic challenges and annotation inconsistencies across databases. All scripts, HMM profiles, and example datasets are available through GitHub, enabling researchers to apply these methods to thiamine pathway analysis or adapt them for other metabolic pathways with similar evolutionary diversity.

Riboswitches are genetic regulatory elements based on RNA molecules that control the expression of genes. Most known metabolite-sensing riboswitches control expression of genes involved in anabolic reactions but a few also control catabolic reactions. In addition, riboswitches control the expression of genes encoding metabolite uptake systems. Binding of a specific small molecule ligand (<1500 Da) to the aptamer ("binder") part of the riboswitch typically results in repression of the downstream genes, in many cases by either transcriptional termination or inhibition of translation. The downstream expression control is supported by the so called "expression platform" which is part of the riboswitch. In this chapter the analysis of thiamine pyrophosphate (TPP)-responsive riboswitches using a specialized dual-luciferase reporter gene assay is exemplarily described and may serve as a blueprint to study other bacterial riboswitches. Also, the mode of action of the riboswitches can be determined by testing transcriptional as well as translational gene fusions using different test plasmids. The effect of candidate ligands which negatively interfere with riboswitch functions can be investigated using this assay, provided the chemicals are taken up by the test organism. The assay can be thus to identify and characterize novel compounds that have the capacity to act as antibiotics. As riboswitches constitute widespread genetic control elements, they represent novel targets for antimicrobial compounds.

Antisense oligonucleotides (ASOs) provide a sequence-specific approach for targeting bacterial RNAs and suppressing the expression of genes important for bacterial growth and proliferation, making them prominent agents in the design of a novel class of antibacterial agents. This protocol describes the design, preparation, and experimental validation of a chimeric ASO conjugated to the cell-penetrating peptide pVEC and directed against the thiamine pyrophosphate (TPP) riboswitch. The workflow includes bioinformatic identification of conserved and accessible regions within the TPP aptamer, design of a chemically modified ASO, preparation of an in vitro transcribed TPP aptamer RNA substrate, and assessment of ASO-dependent RNA cleavage using an RNase H assay. The protocol further describes bacterial growth-inhibition assays in Listeria monocytogenes and Bacillus subtilis, the use of Escherichia coli as a specificity control, and evaluation of pVEC-ASO-1 cytotoxicity in A549 human cells using an MTT-based viability assay. Although presented for the TPP riboswitch, the approach can be adapted to other bacterial mRNA regions. This protocol therefore provides a flexible experimental framework for evaluating riboswitches and other bacterial RNA elements as potential targets for ASO-based antibacterial development.

Cryogenic electron microscopy (cryo-EM) is well suited for studying dynamic macromolecules, but has historically been incompatible with small protein-free RNAs like thiamine pyrophosphate (TPP) riboswitches. This chapter describes a method utilizing an RNA scaffold derived from the Oceanobacillus iheyensis group II intron that overcomes these barriers, allowing for high-resolution reconstructions of an embedded target RNA. This approach produced the first reconstruction of the native open conformation of the apo E. coli TPP riboswitch aptamer, as well as a high-resolution reconstruction of the bound state. The adaptability of the scaffold design allows for any target RNA sequence to easily be embedded for structure determination by cryo-EM. This chapter outlines protocols for construct design parameters, sample preparation, and the unique data processing steps required to use this scaffold to characterize the structures of TPP riboswitch aptamer variants, folding intermediates, and bound to alternate small-molecule aptamer binders.

Biological nanopores equipped with substrate-binding proteins are powerful biosensor pairs capable of measuring low-abundance molecules in complex samples. One example of such a system pairs the thiamine-binding protein (TbpA) and the Cytolysin A (ClyA) nanopore. TbpA has been evolved to selectively bind thiamine in even the most complex samples. When a ClyA nanopore traps TbpA, even a single experiment can generate a vast amount of data in a very short time. While at the research level, even a single nanopore can accurately quantify thiamine concentrations, the setup time prohibits a practical application, such as in point-of-care devices. Such devices would likely house hundreds to millions of pores, rendering manual data analysis impossible. To bridge the gap between biosensor development and practical devices, it is therefore necessary to develop algorithms which can analyze and interpret data automatically. Here we outline our approach to nanopore data analysis to tackle the challenges in automatic analysis.

Biosensing using protein nanopores has become a powerful tool for the analysis of single molecules, enabling for example portable devices for long-read DNA sequencing. By inserting a single pore into a lipid bilayer and applying an electric field, one can create a single-molecule sensor using the ionic current flowing through the pore. Thiamine can be quantified with such a technique using the Cytolysin A pore and a thiamine-binding protein TbpA, which imparts its affinity to the nanopore. When thiamine passes through the pore, it gets captured by TbpA, causing a conformational change that alters the flow of ions through the pore and thus causes a change in current. The result is a biosensor pair with high selectivity that allows quantification of thiamine even in complex solutions. Here, we provide instructions for the implementation of this measurement technique, which includes the production of the required recombinant proteins as well as construction and operation of the electrochemical measuring setup.

Thiazole synthases (THI4s) catalyze the synthesis of the thiazole moiety of thiamin (vitamin B1) from glycine, NAD, and a sulfur atom. The sulfur atom can originate from an active site cysteine residue, as is the case for cysteine-dependent THI4s (cys-THI4s), or from an external sulfide source, as it is for sulfide-dependent THI4s (noncys-THI4s). Cys-THI4s are also known as suicidal enzymes, as catalysis leads to enzyme inactivation, followed by protein degradation. These enzymes can be considered energy intensive and catalytically inefficient enzymes. Replacement of plant cys-THI4s with noncys-THI4s which are not self-inactivating though still slow catalysts, could lead to energy conservation through decreased protein turnover. However, noncys-THI4s are anaerobic or microoxic enzymes and thus would be poor replacements for plant cys-THI4s that are aerobic enzymes. Engineering a plant cys-THI4 to be a true catalyst or a noncys-THI4 to function in plant-like conditions would be necessary to achieve safe respiratory energy. In this chapter, we will provide methods outlining: (1) how to use comparative genomics to select the best candidate THI4s to be engineered via rational design or continuous directed evolution (CDE); (2) how to use rational design to engineer THI4s; and (3) how to use OrthoRep, a yeast-based CDE tool, to engineering noncys-THI4s to function under plant-like conditions.

Thiaminase I enzymes catalyze the cleavage of thiamine (vitamin B1) and have been implicated in dietary and ecological thiamine deficiency syndromes affecting fish, wildlife, livestock, and other organisms. This chapter focuses on the colorimetric 4-nitrothiophenol (4-NTP) assay for thiaminase I activity and discusses alternative techniques. Protocols for sample collection, tissue homogenization, extraction, enzyme preservation, assay optimization, and data analysis are presented in a stepwise format. The application of the assay to bacterial culture supernatants, which offer valuable mechanistic insights at the microbial level, and to more complex tissue homogenates to better understand thiamine deficiency, is discussed. Factors influencing assay performance and interpretation, including endogenous thiamine carryover, endpoint versus kinetic measurements, and physiological relevance of measured in vitro activity, are considered. Emphasis is placed on data processing and normalization. Under the conditions employed, the assay has a limit of detection of 50 pmol 4-NTP degraded per minute, corresponding to 1 ng of thiaminase from Paenibacillus thiaminolyticus. The assay measures maximal catalytic capacity, though we recommend caution when translating thiaminase activities measured under substrate-saturating, elevated-temperature in vitro conditions using homogenized and de-compartmentalized samples to physiological thiamine degradation in vivo. In the latter, thiaminase-mediated thiamine degradation is constrained by substrate availability, an organism's body temperature, biochemical degradation processes, inhibitory factors, and physiological compartmentalization. Collectively, the approach and considerations presented here provide a framework for measuring and interpreting thiaminase I activity in complex biological matrices.

Thiamine diphosphate (ThDP) is a coenzyme for several essential enzymes in cell metabolism (mainly transketolase, pyruvate and 2-oxoglutarate dehydrogenases). In eukaryotic cells ThDP is synthesized by pyrophosphorylation of thiamine (vitamin B1) by ATP or UTP and catalyzed by thiamine pyrophosphokinase isoform 1 (TPK1). Complete inactivation of TPK1 is lethal, but recently several hereditary mutations in the corresponding gene were described in human families. The phenotype is generally severe with acute encephalopathy and fatal outcome when not treated with excess thiamine. This suggests that the mutated enzyme still retains a fraction of its native activity. In order to estimate the effect of the mutations on enzyme activity it is essential to benefit from a reliable assay of TPK1 activity. This enzyme has a low KM for thiamine (KM < 0.1 µM) but a low turnover (kcat = 0.03 s[-1]) requiring very sensitive methods for the detection of the product ThDP by HPLC followed by fluorometric (after derivatization into fluorescent thiochrome derivatives) or mass spectrometric detection. Moreover, ThDP is a noncompetitive inhibitor of TPK1 with a KI = 0.4 µM. Hence, when measuring TPK1 activity care must be taken that the reaction is stopped before inhibition becomes significant. Here, we present a powerful HPLC method coupled to fluorometric detection allowing TPK1 assay either in the forward or the reverse direction. The importance of such a method is further highlighted by recent data showing decreased expression of TPK1 in blood and brain of Alzheimer patients making TPK1 assay an interesting alternative to the estimation of TPK1 expression.

Thiamine (vitamin B1) as thiamine diphosphate (or thiamine pyrophosphate) is a cofactor for enzymes involved in energy metabolism and is essential for brain function. Reliable biomarkers of thiamine status and function are necessary to understand thiamine's role in health and disease and provide mechanistic insights and to determine the efficacy of pharmacological interventions of thiamine analogues. The activity of the thiamine-dependent transketolase enzyme in erythrocytes is the established functional marker of whole-body thiamine status. In this chapter, we describe the analogous method optimized for the determination of transketolase activity in brain tissue and the calculation of the brain transketolase 'activity coefficient'. Provided are full method details for the preparation of brain material and instructions to perform the assay in 96-well plates using a microplate spectrophotometer. We provide details of suitable quality control materials and tips for troubleshooting. Instructions are also provided for the calculation of absolute basal transketolase activity. These protocols provide standardization for the determination of transketolase activity in human post mortem brain samples or from animal experiments and will be useful for researchers interested in the role of thiamine in brain-related conditions. Such protocols are essential to harmonize methods between laboratories for the benefit of comparisons between studies.

Thiamine (vitamin B1) is an essential nutrient with established adverse health consequences associated with acute deficiency. Emerging evidence also suggests long-term neurocognitive deficits due to sub-clinical deficiency in early life, and there is a growing appreciation of a potential role for thiamine in Alzheimer's disease. Therefore, the assessment of thiamine biomarkers, particularly in large, population-based studies, is critical to grasp the true scope of this public health problem. The most reliable biomarkers of thiamine status are whole blood concentrations of thiamine diphosphate (ThDP) measured by high performance liquid chromatography (HPLC) with fluorescence detection or liquid chromatography tandem mass spectrometry (LC-MS/MS), and the functional assay of transketolase activity (erythrocyte transketolase activity coefficient, ETKAC). Plasma thiamine reflects recent intake, and is not a useful biomarker of thiamine status. This manuscript describes the different biomarkers and methods of assessment, their advantages and disadvantages, and related cutoffs for deficiency. A table of 'performance characteristics' is provided to guide users on the quality and performance characteristics of each assay, and practicalities related to their cost, resource requirement, and maintenance. Provision of these insights will help inform decisions about inclusion of thiamine analysis in future work to better understand the extent of global thiamine deficiency.