Metabolic engineering provides insight into the regulation of thiamin biosynthesis in plants

Abstract

Thiamin (or thiamine) is a water-soluble B-vitamin (B1), which is required, in the form of thiamin pyrophosphate (TPP), as an essential cofactor in crucial carbon metabolism reactions in all forms of life. To ensure adequate metabolic functioning, humans rely on a sufficient dietary supply of thiamin. Increasing thiamin levels in plants via metabolic engineering is a powerful strategy to alleviate vitamin B1 malnutrition and thus improve global human health. These engineering strategies rely on comprehensive knowledge of plant thiamin metabolism and its regulation. Here, multiple metabolic engineering strategies were examined in the model plant Arabidopsis thaliana. This was achieved by constitutive overexpression of the three biosynthesis genes responsible for B1 synthesis, HMP-P synthase (THIC), HET-P synthase (THI1) and HMP-P kinase/TMP pyrophosphorylase (TH1), either separate or in combination. By monitoring the levels of thiamin, its phosphorylated entities, and its biosynthetic intermediates, we gained insight into the effect of either strategy on thiamin biosynthesis. Moreover, expression analysis of thiamin biosynthesis genes showed the plant’s intriguing ability to respond to alterations in the pathway. Overall, we revealed the necessity to balance the pyrimidine and thiazole branches of thiamin biosynthesis and assessed its biosynthetic intermediates. Furthermore, the accumulation of non-phosphorylated intermediates demonstrated the inefficiency of endogenous thiamin salvage mechanisms. These results serve as guidelines in the development of novel thiamin metabolic engineering strategies.

A novel panel of yeast assays for the assessment of thiamin and its biosynthetic intermediates in plant tissues

“…Therefore, a quick scan of engineered lines to assess adequate metabolic flux towards the desired products, whilst limiting the build‐up of unwanted intermediates can be advantageous. Examples of such studies include the enhancement of vitB1 in Arabidopsis (Bocobza et al ., 2013; Dong et al ., 2015; Strobbe et al ., 2021b), rice (Dong et al ., 2016; Strobbe et al ., 2021a) and Lotus japonicus (Yin et al ., 2019). In some cases, high levels of these metabolites are to be avoided since not all compounds are generally regarded as safe (GRAS (generally recognized as safe) compounds).…”

“…By contrast, when considering biofortification through breeding, changes may be more subtle due to the limited natural variation in thiamin and thiamin‐related metabolites. For these purposes, yeast microbiological assays will likely only be useful for the assessment of total vitB1, as the intermediates can be overshadowed since these metabolites typically only reach a fraction of the molar total vitB1 concentration (Strobbe et al ., 2021a,b). Breeding of crops towards higher vitamin content has shown to be a successful method to ensure higher vitamin consumption by certain populations (Bouis & Saltzman, 2017).…”

“…Note that the methodology is not able to distinguish between HMP‐P and HMP‐PP; their sum is measured as phosphorylated HMP. The method was fully validated according to international guidelines (Verstraete et al ., 2020), and has shown its applicability in characterization of engineered transgenic Arabidopsis lines (Strobbe et al ., 2021b).…”

“…Recently, we described a quantitative liquid chromatography–tandem mass spectrometry (LC‐MS/MS) method for the determination of thiamin, its biosynthetic intermediates (4‐amino‐2‐methyl‐5‐hydroxymethylpyrimidine (HMP) and 4‐methyl‐5‐(2‐hydroxyethyl) thiazole (HET)) and phosphate derivatives (TMP and TPP) in Arabidopsis thaliana (Verstraete et al ., 2020) and in rice ( Oryza sativa ) (Verstraete et al ., 2021). As A. thaliana serves as a reference plant for plant genetics, the plant material is perfectly suited to perform preliminary genetic research to gain new insights into (regulation of) thiamin biosynthesis (Dong et al ., 2015; Noordally et al ., 2020; Strobbe et al ., 2021b). Although the aforementioned HPLC and LC–MS/MS methods are highly sensitive, specific and precise, an extensive sample preparation procedure is required (Martinis et al ., 2016; Verstraete et al ., 2020).…”

“…The reasoning behind this is two‐fold. First, assessment of intermediate accumulation can provide information to identify metabolic hurdles, which could be obstructing satisfactory biosynthetic flux towards the end product (Schaub et al ., 2005; Blancquaert et al ., 2013; Pourcel et al ., 2013; Strobbe et al ., 2021b). Second, high accumulation of intermediates, occurring as an undesired side effect of the metabolic engineering strategy, should be monitored and limited (Schaub et al ., 2005; Blancquaert et al ., 2013; Pourcel et al ., 2013; Strobbe et al ., 2021b).…”