Evaluation of Brachypodium distachyon L-Tyrosine Decarboxylase Using L-Tyrosine Over-Producing Saccharomyces cerevisiae

Abstract

To demonstrate that herbaceous biomass is a versatile gene resource, we focused on the model plant Brachypodium distachyon, and screened the B. distachyon for homologs of tyrosine decarboxylase (TDC), which is involved in the modification of aromatic compounds. A total of 5 candidate genes were identified in cDNA libraries of B. distachyon and were introduced into Saccharomyces cerevisiae to evaluate TDC expression and tyramine production. It is suggested that two TDCs encoded in the transcripts Bradi2g51120.1 and Bradi2g51170.1 have L-tyrosine decarboxylation activity. Bradi2g51170.1 was introduced into the L-tyrosine over-producing strain of S. cerevisiae that was constructed by the introduction of mutant genes that promote deregulated feedback inhibition. The amount of tyramine produced by the resulting transformant was 6.6-fold higher (approximately 200 mg/L) than the control strain, indicating that B. distachyon TDC effectively converts L-tyrosine to tyramine. Our results suggest that B. distachyon possesses enzymes that are capable of modifying aromatic residues, and that S. cerevisiae is a suitable host for the production of L-tyrosine derivatives.

Fig 1. Proposed biosynthesis pathway for tyramine (ARO3, ARO4; 3-deoxy-D-heptulosonate-7-phosphate synthase: ARO7; chorismate mutase: TDC; L-tyrosine decarboxylase).

ARO3, ARO4 and ARO7 are derived from S. cerevisiae, whereas TDC is originated from B. distachyon.

Fig 2. Evaluation of L-tyrosine over-producing S. cerevisiae constructed in this study.

Each bar chart shows the average of 3 independent experiments, and error bars represent the standard deviation. (A) Evaluation of L-tyrosine productivity in the culture supernatants of YPH499/δU/δL, YPH499/δUARO4 ^fbr/δL, YPH499/δU/δLARO7 ^fbr, and YPH499/δUARO4 ^fbr/δLARO7 ^fbr. (B) Determination of ARO7 and ARO7 ^fbr gene copy numbers in YPH499/δU/δL, YPH499/δUARO4 ^fbr/δL, YPH499/δU/δLARO7 ^fbr, and YPH499/δUARO4 ^fbr/δLARO7 ^fbr (ARO4 ^fbr; Ser to Ala substitution in ARO4 at position 195: Gly to Ser substitution in ARO7 at position 141).

Fig 3. Culture profiles of transformants in SD medium containing 2% glucose as the carbon source.

Time-courses of (A) cell growth, (B) glucose consumption, (C) ethanol production, and (D) tyramine production for YPH499/δU/δL/tdc70 (crosses), YPH499/δU/δLARO7 ^fbr/tdc70 (triangles), YPH499/δUARO4 ^fbr/δL/tdc70 (squares), and YPH499/δUARO4 ^fbr/δLARO7 ^fbr/tdc70 (circles). Each data point shows the average of 3 independent experiments, and error bars represent the standard deviation.