*Regulation of Thiamin Biosynthesis*

THIC promoter has been identified to be essential for the circadian clock-regulation of thiamin biosynthesis [25]. Thiamin levels are also regulated by a TPP responsive riboswitch located at the 3' region of the THIC pre-mRNA, controlling the stability of THIC mRNA [25]. Riboswitches are mRNA sensors that bind small molecules and consecutively regulate gene expression. The TPP riboswitch mediates a feedback regulation of the thiamin biosynthesis pathway. When TPP levels are high, TPP binds to the riboswitch leading to intron splicing and instable THIC mRNA, which is consequently degraded [24,117].

Another layer of regulation relies on the activities of thiamin biosynthesis enzymes. THI1 is a single turnover protein; it loses functionality once the cysteine residue is used in the reaction [107,118], and thus a single step of thiamin biosynthesis requires high energy levels. In addition, excess HMP-PP could work as an inhibitor for TH1 activity, as suggested by *THIC*-overexpressing plants [119]. Bioengineering studies for the fortification of crops also showed that an increase in expression of *THIC* and *THI1* in Arabidopsis and rice plants does not necessarily result in an increase in thiamin production [112,114,120]; these enzymes rely on factors, such as reduction by thioredoxins for THIC and THI1, and supply of sulfur for THIC's iron-sulfur cluster [104,114,121], which could also be limiting.

**Figure 3.** Thiamin biosynthesis pathway in plants. Thiamin is synthesized from pyrimidine and a thiazole moiety, both of which are synthesized in the chloroplast. The detailed description of the pathway is presented in the text. Abbreviations of the substrates: 5-aminoimidazole ribonucleotide (AIR), S-adenosylmethionine (SAM), nicotinamide adenine dinucleotide (NAD+), 4-amino-2-methyl-5-hydroxymethylpyrimidine phosphate (HMP-P), adenylated thiazole intermediate (ADT), 4-methyl-5-b-hydroxyethylthiazole phosphate (HET-P), thiamin monophosphate (TMP), thiamin pyrophosphate (TPP). Abbreviations of the enzymes: thiamin C synthase (THIC), -methyl-5-b-hydroxyethylthiazole phosphate (HET-P) synthase (THI1), thiamin monophosphate pyrophosphorylase (TH1), haloacid dehalogenase (HAD) family phosphatase (TH2), TPP kinases (TDPK).

The complexity of thiamin biosynthesis regulation and the universality of thiamin-requiring enzymes across kingdoms and their association with core metabolic pathways suggest a crucial role of TPP in the regulation of cellular metabolism and the potentially prejudicial effect of inappropriate total thiamin levels. The cell localization of the main thiamine-dependent enzymes and its participation in metabolic pathways are depicted in Figure 4.

**Figure 4.** Cell localization of the main thiamin dependent-enzymes and its participation in metabolic pathways. Thiamin pyrophosphate (TPP), the active form of thiamin, works as an essential coenzyme for the enzymes involved in photosynthesis in chloroplasts, pentose phosphate pathway, and alcoholic fermentation in cytoplasm, as well as in ATP synthesis in the participation in oxidative decarboxylation of pyruvate and tricarboxylic acid cycle in mitochondrial central metabolism. Thiamin has also been shown to be involved in the acclimation responses to abiotic stresses; photoperiod; and working directly as an antioxidant, scavenging ROS; a protection molecule; and indirectly by contributing to the cell energy poll, conferring the cell the necessary metabolic flexibility to acclimate to new conditions. The thiamin-dependent enzymes shown are α-ketose transketolase (TK); 1-deoxy-D-xylulose-5-phosphate synthase (DXPS); acetohydroxyacid synthase (AHAS); pyruvate dehydrogenase (PDH); 2-oxoglutarate dehydrogenase (OGDH); and branched chain 2-oxoacid dehydrogenase (BCOADH). MEP, methylerythritol pathway.
