Changes in Primary and Secondary Metabolite Levels in Response to Gene Targeting-Mediated Site-Directed Mutagenesis of the Anthranilate Synthase Gene in Rice

Abstract

Gene targeting (GT) via homologous recombination allows precise modification of a target gene of interest. In a previous study, we successfully used GT to produce rice plants accumulating high levels of free tryptophan (Trp) in mature seeds and young leaves via targeted modification of a gene encoding anthranilate synthase—a key enzyme of Trp biosynthesis. Here, we performed metabolome analysis in the leaves and mature seeds of GT plants. Of 72 metabolites detected in both organs, a total of 13, including Trp, involved in amino acid metabolism, accumulated to levels >1.5-fold higher than controls in both leaves and mature seeds of GT plants. Surprisingly, the contents of certain metabolites valuable for both humans and livestock, such as γ-aminobutyric acid and vitamin B, were significantly increased in mature seeds of GT plants. Moreover, untargeted analysis using LC-MS revealed that secondary metabolites, including an indole alkaloid, 2-[2-hydroxy-3-β-D-glucopyranosyloxy-1-(1H-indol-3-yl)propyl] tryptophan, also accumulate to higher levels in GT plants. Some of these metabolite changes in plants produced via GT are similar to those observed in plants over expressing mutated genes, thus demonstrating that in vivo protein engineering via GT can be an effective approach to metabolic engineering in crops.

TrpE feedback mutants reveal roadblocks and conduits toward increasing secondary metabolism in Aspergillus fumigatus

“…Two mutant alleles were created, one was designed to change serine residue 77 to leucine (S77L), and the other was designed to additionally change serine residue 66 to arginine (S66R). These residues were chosen as they have been shown essential for tryptophan feedback inhibition in yeast (Graf et al, 1993) and some plants (Kanno et al, 2005; Saika et al, 2012). Transformation of the Δ trpE strain with a wild-type and these two mutant trpE alleles, respectively, yielded three strains TPMW8.9 ( trpE C ), TPMW9.5 ( trpE S77L ) and TPMW10.9 ( trpE S66R,S77L ) for comparison of aromatic acid metabolism and Fq synthesis (Fig.…”

“…The importance of the feedback regulation of AS in the control of metabolic flow in l -tryptophan production and secondary metabolites has been demonstrated in many plants expressing feedback-resistant AS genes (Dubouzet et al, 2013; Hong et al, 2006; Hughes et al, 2004; Ishihara et al, 2006; Li and Last, 1996; Saika et al, 2012; Tozawa et al, 2001). Here, inactivation of the putative feedback domain in TrpE of A. fumigatus also had a significant effect on aromatic amino acid metabolism and Fq production with the single mutation strain trpE S77L consistently having a greater impact on metabolism than the double site mutant.…”

“…ASs have been characterized in bacteria (Bae et al, 1989; Ito et al, 1969; Palmer, G.C. et al, 2013), in plants (Morino et al, 2005; Saika et al, 2012; Tozawa et al, 2001) and in Saccharomyces cerevisiae (Braus, 1991; Graf et al, 1993). In yeast, AS consists of two subunits (AAS-I and AAS-II) that are necessary to yield anthranilate from chorismate by addition of an amino group from l -glutamine (Graf et al, 1993).…”