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 Caladium bicolor that enables production and emission of large amounts of the cThz-P analog 4-methyl-5-vinylthiazole (MVT). We used functional complementation of an Escherichia coli ΔthiG strain to identify a nonsuicidal bacterial THI4 (from Thermovibrio ammonificans) that can function in conditions like those in plant cells. We explored whether C. bicolor 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 C. bicolor flowers make MVT de novo via a massively expressed THI4 and that thiamin is not involved. Functional complementation tests indicated that C. bicolor THI4, which has the active-site Cys needed to operate suicidally, may be capable of suicidal and - in hypoxic conditions - nonsuicidal operation. T. ammonificans and C. bicolor THI4s are thus candidate parts for rational redesign or directed evolution of efficient, nonsuicidal THI4s for use in crop improvement.

Figure 1.

THI4-mediated synthesis of the thiamin thiazole precursors and their possible relationship to MVT. A, In the plant thiazole synthesis pathway, THI4 forms the adenylated thiazole precursor ADT from NAD⁺, Gly, and a sulfur atom stripped from an active-site Cys residue, which is changed to dehydro-Ala (DHAla). The inactive DHAla form of THI4 is degraded. ADT is cleaved to give cThz-P, the direct precursor of thiamin. MVT could theoretically be derived from ADT or cThz-P by decarboxylation and elimination of ADP or phosphate, respectively (hypothetical reactions shown in blue). B, In thiazole synthesis in Methanococci, sulfide (as HS^-) serves as the sulfur donor, and the THI4 enzyme is not inactivated in the reaction. The pathway is otherwise the same as in plants. C, Partial sequence alignment showing the active site Cys in Arabidopsis THI4 and its replacement by His in Methanococcus jannaschii THI4.

Figure 2.

Tests of functional complementation of an E. coli ΔthiG strain by diverse bacterial THI4s. A, The ΔthiG strain harboring the pBAD24 vector alone or containing a THI4 gene was cultured in MOPS minimal medium containing 0.2% (w/v) glycerol and 0.02% (w/v) arabinose, plus or minus 1 mm Cys, in aerobic or anaerobic conditions. Aerobic controls supplemented with 100 nm thiamin were included. Anaerobic media contained 40 mm nitrate as the electron acceptor. Overnight liquid cultures of three independent clones for each construct were 10-fold serially diluted and spotted on the plates. Images were captured after incubation at 37°C for 7 d. B, Growth in liquid MOPS medium as above, plus 1 mm Cys, of wild type E. coli (WT) and the ΔthiG strain harboring pBAD24 alone (EV) or containing T. ammonificans THI4 (TaTHI4). Cultures were incubated in aerobic conditions at 37°C. C, Thiamin monophosphate (ThMP) and diphosphate (ThDP) contents of wild type and TaTHI4-complemented cells from B, harvested when OD₆₀₀ reached 1.0–1.5; free thiamin was undetectable. Values in B and C are means and SE for three independent replicates. The difference in ThDP content between wild type and TaTHI4-complemented cells was significant at P < 0.001 (**), as determined by Student’s t test. Where no error bars appear in B they are smaller than the symbol.

Figure 3.

MVT emission from C. bicolor inflorescences. A, Inflorescence dissection showing the parts tested for emissions. The female, sterile male, and fertile male flowers together comprise the spadix. B, MVT emission from inflorescence parts of cv ‘Pink Gem’. Data are means and SE for two biological replicates. Volatiles were collected for 2 h (19:30-21:30). C, Hourly MVT emission rates from sterile and fertile male flower tissue of cv ‘Tapestry’. Data are means and SE for three biological replicates. The x axis shows the start time for each 1-h collection period.

Figure 4.

Developmental time courses of the levels of extractable MVT (A) and of thiamin and its phosphorylated forms (B) in male flower tissue of cv ‘Tapestry’. Data are means and SE of three biological replicates. Tissue was harvested 1, 3, and 10 d before female anthesis, on day 0 (female anthesis), and 1 d after female anthesis. The day 0 harvest was at 20:30, when the MVT emission burst was in progress. ThDP, thiamin diphosphate; ThMP, thiamin monophosphate.

Figure 5.

RT-qPCR assay of THI4 mRNA levels in various organs of C. bicolor cv ‘Tapestry’. THI4 mRNA was quantified by the 2^-ΔΔCT method. Data are presented as the -fold difference in THI4 gene expression in each organ normalized to the reference gene (actin or 18S rRNA) and relative to roots (= 1.0). Inflorescence tissues were harvested on Day 0, just before the MVT emission burst started. Data are means and SE of three biological replicates. Note that the y axis scale is logarithmic.

Figure 6.

Functional complementation of the E. coli ΔthiG strain by native and C228A mutant C. bicolor THI4. A, The ΔthiG strain harboring the pBAD24 vector alone or containing the native or C228A mutant form of C. bicolor THI4_1 was cultured in MOPS minimal medium containing 0.2% (w/v) glycerol and 0.02% (w/v) arabinose, plus or minus 1 mm Cys, in aerobic or anaerobic conditions. Controls supplemented with 100 nm thiamin were included. Anaerobic media contained 40 mm nitrate as the electron acceptor. Overnight liquid cultures of three independent clones for each construct were 10-fold serially diluted and spotted on the plates. Images were captured after incubation at 37°C for 7 d. B, Thiamin monophosphate (ThMP) and diphosphate (ThDP) contents (log scale) of E. coli ΔthiG cells complemented with native C. bicolor THI4_1, grown aerobically without added Cys; wild type cells grown in these conditions are included for comparison. C, Thiamin mono- and diphosphate contents (log scale) of E. coli ΔthiG cells complemented with native or C228A C. bicolor THI4_1, grown anaerobically plus 1 mm Cys; wild type cells grown in these conditions are included for comparison. Values in B and C are means and SE for three independent replicates. No thiamin monophosphate was detectable in cells complemented with native or C228A C. bicolor THI4_1. The ThDP content differences between wild type and complemented cells were significant at P < 0.0001 (***) in aerobic conditions or P < 0.05 (*) in anaerobic conditions, as determined by Student’s t test.