Parts-Prospecting for a High-Efficiency Thiamin Thiazole Biosynthesis Pathway

Area of Science:

• Biochemistry
• Plant Science
• Metabolic Engineering

Background:

• Plants synthesize thiamin's thiazole precursor (cThz-P) using a suicidal THIAMIN4 (THI4) enzyme.
• This THI4 enzyme's turnover is energy-intensive, consuming 2-12% of a plant's energy budget.
• Alternative, non-suicidal THI4 pathways exist in prokaryotes and potentially in *Caladium bicolor*.

Purpose of the Study:

• To identify and characterize non-suicidal THI4 enzymes for potential crop improvement.
• To investigate the mechanism of 4-methyl-5-vinylthiazole (MVT) synthesis in *Caladium bicolor*.
• To assess the feasibility of using alternative THI4 pathways to enhance crop yield potential.

Main Methods:

• Functional complementation of an *Escherichia coli* Δ*thiG* strain to test bacterial THI4 activity.
• Analysis of MVT emission, extractable MVT, thiamin levels, and THI4 gene expression in *C. bicolor* flowers.
• Biochemical assays to determine the suicidal or non-suicidal activity of *C. bicolor* THI4 under varying conditions.

Main Results:

• A non-suicidal THI4 from *Thermovibrio ammonificans* was identified and shown to function in plant-like conditions.
• *Caladium bicolor* flowers synthesize MVT de novo via a highly expressed THI4, independent of thiamin.
• *C. bicolor* THI4 exhibits potential for both suicidal and non-suicidal activity, particularly under hypoxia.

Conclusions:

• *T. ammonificans* and *C. bicolor* THI4 enzymes are promising candidates for engineering more energy-efficient thiamin synthesis pathways.
• Developing non-suicidal THI4s could lead to substantial increases in crop yield potential.
• Understanding plant thiamin biosynthesis offers avenues for metabolic engineering in agriculture.