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

Metabolic engineering of thiamin (vitamin B1) content emphasizes the need to balance the two branches of B1 biosynthesis and uncovers the importance of B1 salvage to improve engineering strategies.


Supplemental figure S2. Schematic representation of several primers used in reverse transcription quantitative PCR reactions.
3'UTR's are indicated with gray arrows, while 5'UTR regions are indicated with a small gray block. Coding sequences (CDS) are represented by colored arrows (length of the arrow is relative to CDS length). A Different primer pairs were designed to investigate transcript levels of both native and transgenic transcript abundance of THIC and THI1 in quantitative real-time PCR (qRT-PCR) analysis. Primers inside the CDS of the genes allowed quantification of both native and transgenic transcript levels. This is accomplished by pairs P1-P2 and P5-P6 for THIC and THI1 expression, respectively. Unless specifically stated (as 'transgenic' expression), this total expression, meaning the sum of both native and transgenic expression is intended. Utilization of primers spanning the CDS-3'UTR border, which discriminates between native and transgenic transcripts, allows insight in the abundance of transgenic transcript upon their utilization in qRT-PCR analysis. This is accomplished by utilization of primer pairs P3-P4 and P7-P4 to allow insights in accumulation of transgenic THIC and THI1 transcripts, respectively. B Designing of a primer pair in a specific region inside the CDS conserved among TPK1 and TPK2, allows amplification of an (almost) identical transcript (equal length) and thereby allows quantification of total TPK transcript abundance (sum of TPK1 and TPK2). Black zones inside the CDS represent conserved regions, while red zones indicate minute differences between TPK1 and TPK2. Gene information. THIC, At2g29630, 1935 bp; THI1, At5g54770, 1050 bp; TPK1, At1g02880, 804 bp, TPK2, At2g44750, 804 bp.

Supplemental figure S3. Relative abundance of metabolites in the WT and engineered Arabidopsis lines.
Left, piecharts representing the relative molar abundance of different B1 vitamers (thiamin, TMP and TPP) in WT and transgenic lines. Right, pie-charts representing the relative molar abundance of metabolites committed towards B1 biosynthesis (total HMP, total HET, thiamin, TMP and TPP).

Supplemental figure S4. Correlation between engineered gene expression and metabolite levels of the corresponding intermediate in THIC-and THI1-engineered lines.
In THIC-engineered lines, THIC expression correlated with accumulation of total HMP (A) as well as non-phosphorylated HMP (B). In THI1-engineered lines, THI1 expression correlated with accumulation of total HET (C) as well as non-phosphorylated HET (D). The specific engineered lines depicted in the graphs are given a number code (E). Data points represent values for both metabolite content and expression levels, as displayed in Fig 3A and 3B. Logarithmic approximation of expression/metabolite prediction as well as correlation is displayed as calculated using Microsoft Excel. Interpretation. As the supply of the thiazole intermediate is potentially limiting B1 accumulation, there is a need to ameliorate metabolic engineering approaches to boost production of this metabolite. The high molar quantities of pyrimidine produced upon THIC overexpression, together with the potential toxicity of accumulating pyrimidine metabolites, indicates the need to balance THIC expression with thiazole production. This could be achieved by increasing both THIC and THI1 expression to a level sufficient to provide an additional 2 nmol/g supply of pyrimidine and thiazole intermediates, respectively. Based on the observed correlation between gene expression and the accumulation of the corresponding intermediates, one can calculate that such levels could theoretically be reached with a 2-fold increase in THIC expression combined with 7-fold increase in THI1 expression. The observation that the enhancement in thiamin achieved in the most successful lines (e.g. TTT-2.2.1, TTT-44.5) (see Fig. 4B), is in the same order of magnitude as the increment in the thiazole intermediate in THI1-engineered lines (up to 2 nmol/g FW, see Fig. 3B) further emphasizes the rate-limiting role to thiazole production. S5. Phenotype of all soil-grown engineered lines. All lines were grown on soil (16/8 light/dark regime; 21 °C; 50% relative humidity; 150μmol m -2 S -1 white light) and photographed at 30 days of age. Rosettes were digitally extracted to allow comparison. White bars indicate 1 cm, all images including different lines, are set to the same scale. Designation of the line is indicated above the depicted plant. An aberrant phenotype was witnessed in THIC overexpressing lines, with the exception of line THIC-37.8, which depicted modest THIC overexpression (THI1 and TH1engineered lines resembled WT). The typical phenotype included chlorosis and exaggerated leaf serrations.

Supplemental figure S6. Analysis of metabolite levels in a selection of soil-grown Arabidopsis plants.
Metabolite levels were measured via LC-MS/MS in Arabidopsis plants grown on soil for 30 days (Fig. S5). Samples are mean (±SE) of 3 biological repeats, each consisting of complete leaf tissues from multiple individual plants. Statistical difference compared to WT is indicated to be significant (single asterisk, p <0.05) or very significant (double asterisks, p <0.01).

Supplemental figure S7. Estimation of thiamin biosynthesis gene transcript levels in WT Arabidopsis.
Relative (endogenous) gene expression of thiamin biosynthesis genes THIC, THI1, TH1, TH2 and TPK1/TPK2 in WT is depicted, as revealed by real-time quantitative PCR (qRT-PCR). Values are semi-quantitative, as they are based on qRT-PCR threshold (Ct) values using different primer sets (cT values: THIC, 22.5; THI1, 17; TH1, 29.5; TH2, 23.5; TPK, 22.5). As all primer pairs are optimized to have adequate efficiency (95-105%), comparison of their raw Ct values, allows relative estimation of transcript abundance. Values for 'TPK' comprise combined expression of TPK1 and TPK2 (primers target an amplicon in conserved region). Relative transcript levels of thiamin biosynthesis genes are depicted in which TH1 expression is set to be 1 (values are relative to TH1). Data was collected from 15-day old seedlings grown on half-strength Murashige and Skoog medium supplemented with 10g/liter sucrose. Samples were collected in the middle of the light period. Values are means of three independent biological repeats (± standard error). Data analysis and normalization were performed using the qBASE software, based on the 2 -ΔΔCt method (Livak and Schmittgen, 2001;Hellemans et al., 2007) Specific information regarding primers and conditions can be retrieved from supplemental table S1.
Supplemental table 1. Information regarding the different primer pairs. Gateway cloning attB sequences are indicated in bold and italics. Gene specific parts in gateway cloning are underlined. Primer numbers of primers depicted in Fig. S2 are indicated in bold.