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.

Bioinformatic and experimental evidence for suicidal and catalytic plant THI4s

“…pET28b constructs were introduced into an E. coli BL21(DE3) ΔthiG strain constructed by recombineering [6,26]. Three independent clones of each construct were used to inoculate 3 ml of MOPS medium [27] containing 0.2% glucose, 100 nM thiamin, and 50 μg/ml kanamycin.…”

“…After incubation at 37°C for 18 h, cells were harvested by centrifugation, washed five times with thiamin-free MOPS medium, resuspended in 500 μl of the same medium, serially diluted in ten-fold steps, and spotted on MOPS medium plates containing 0.2% glucose, 1 mM IPTG, plus or minus 1 mM Cys. Plates were then incubated at 37°C in aerobic conditions as described [6]. After 6 d (or 2 d for Arabidopsis), images were captured and digitized to quantify cell growth using ImageJ software.…”

“…First, the active-site Cys is conserved in all plant THI4 sequences examined so far [5,6]. Second, the active-site Cys residue in arum lily THI4 was shown to be required for functional complementation of an Escherichia coli thiazole auxotroph [6]. Third, when Arabidopsis THI4 was expressed in E. coli the active-site Cys was apparently converted to DHAla [1,7].…”

“…Indirect evidence implies that plant THI4s, like yeast THI4, are suicide enzymes. First, the active-site Cys is conserved in all plant THI4 sequences examined so far [5,6]. Second, the active-site Cys residue in arum lily THI4 was shown to be required for functional complementation of an Escherichia coli thiazole auxotroph [6].…”

“…(Henceforth, we term these Cys and non-Cys THI4s, respectively.) This exploration was prompted by the recent findings (i) that non-Cys THI4s occur not only in anaerobic prokaryotes from sulfide-rich environments but also in aerotolerant and microaerophilic -or possibly aerobic -organisms from low-sulfide environments, and (ii) that some of these THI4s can function in E. coli in the presence of oxygen [6]. These findings suggested that catalytic THI4s might be able to operate in the conditions found in plant cells, i.e.…”