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

Abstract

Plants synthesize the thiazole precursor of thiamin (cThz-P) via THIAMIN4 (THI4), a suicide enzyme that mediates one reaction cycle and must then be degraded and resynthesized. It has been estimated that this THI4 turnover consumes 2% to 12% of the maintenance energy budget and that installing an energy-efficient alternative pathway could substantially increase crop yield potential. Available data point to two natural alternatives to the suicidal THI4 pathway: (i) nonsuicidal prokaryotic THI4s that lack the active-site Cys residue on which suicide activity depends, and (ii) an uncharacterized thiazole synthesis pathway in flowers of the tropical arum lily <i>Caladium bicolor</i> that enables production and emission of large amounts of the cThz-P analog 4-methyl-5-vinylthiazole (MVT). We used functional complementation of an <i>Escherichia coli</i> Δ<i>thiG</i> strain to identify a nonsuicidal bacterial THI4 (from <i>Thermovibrio ammonificans</i>) that can function in conditions like those in plant cells. We explored whether <i>C. bicolor</i> synthesizes MVT de novo via a novel route, via a suicidal or a nonsuicidal THI4, or by catabolizing thiamin. Analysis of developmental changes in MVT emission, extractable MVT, thiamin level, and THI4 expression indicated that <i>C. bicolor</i> flowers make MVT de novo via a massively expressed THI4 and that thiamin is not involved. Functional complementation tests indicated that <i>C. bicolor</i> THI4, which has the active-site Cys needed to operate suicidally, may be capable of suicidal and - in hypoxic conditions - nonsuicidal operation. <i>T. ammonificans</i> and <i>C. bicolor</i> THI4s are thus candidate parts for rational redesign or directed evolution of efficient, nonsuicidal THI4s for use in crop improvement.