Characterization of the Formation of Branched Short-Chain Fatty Acid:CoAs for Bitter Acid Biosynthesis in Hop Glandular Trichomes

Abstract

Bitter acids, known for their use as beer flavoring and for their diverse biological activities, are predominantly formed in hop (Humulus lupulus) glandular trichomes. Branched short-chain acyl-CoAs (e.g. isobutyryl-CoA, isovaleryl-CoA and 2-methylbutyryl-CoA), derived from the degradation of branched-chain amino acids (BCAAs), are essential building blocks for the biosynthesis of bitter acids in hops. However, little is known regarding what components are needed to produce and maintain the pool of branched short-chain acyl-CoAs in hop trichomes. Here, we present several lines of evidence that both CoA ligases and thioesterases are likely involved in bitter acid biosynthesis. Recombinant HlCCL2 (carboxyl CoA ligase) protein had high specific activity for isovaleric acid as a substrate (K cat /K m = 4100 s(-1) M(-1)), whereas recombinant HlCCL4 specifically utilized isobutyric acid (Kcat/K m = 1800 s(-1) M(-1)) and 2-methylbutyric acid (Kcat/K m = 6900 s(-1) M(-1)) as substrates. Both HlCCLs, like hop valerophenone synthase (HlVPS), were expressed strongly in glandular trichomes and localized to the cytoplasm. Co-expression of HlCCL2 and HlCCL4 with HlVPS in yeast led to significant production of acylphloroglucinols (the direct precursors for bitter acid biosynthesis), which further confirmed the biochemical function of these two HlCCLs in vivo. Functional identification of a thioesterase that catalyzed the reverse reaction of CCLs in mitochondria, together with the comprehensive analysis of genes involved BCAA catabolism, supported the idea that cytosolic CoA ligases are required for linking BCAA degradation and bitter acid biosynthesis in glandular trichomes. The evolution and other possible physiological roles of branched short-chain fatty acid:CoA ligases in planta are also discussed.

Nicotinate O-Glucosylation Is an Evolutionarily Metabolic Trait Important for Seed Germination under Stress Conditions in Arabidopsis thaliana

“…The protein expression constructs of the Arabidopsis GT genes were generated using a previously described strategy (Xu et al, 2013). Twelve NANGT candidate genes (AT1G10400, AT1G64910, AT2G15490, AT2G22930, AT2G29750, AT3G02100, AT3G46690, AT5G05860, AT5G05870, AT5G05880, AT5G05890, and AT5G05900) and eight UGT74F2 homolog genes (AT1G05530, AT1G05560, AT1G05680, AT1G24100, AT2G31790, AT2G31750, AT2G43820, and AT2G43840) were subcloned into the pEXP5-CT TOPO vector (Invitrogen) or pMAL-c2x vector (New England Biolabs) (see Supplemental Data Set 3 for primer information).…”

Recent progress in secondary metabolism of plant glandular trichomes

“…However, the routes to the CoA-activated substrates in hops trichomes have been overlooked. Recently we extensively studied the upstream branches of the bitter acid biosynthetic pathway, and found that the mitochondrial thioesterase genes (HlTE4) and cytosolic branched short chain fatty acid CoA ligase genes (HlCCL2 and HlCCL4) play critical roles in linking the BCAA (branched chain amino acid) degradation pathway and the bitter acid pathway (Xu et al 2013).…”

“…This engineered yeast also offers a bio-platform to further characterize the remaining enzymatic steps in the bitter acid biosynthesis pathway, e.g. the prenyltransferase and monooxygenase catalyzed reactions (Xu et al 2013). Following the outline in Figure 4, we will generate a yeast strain producing bitter acid/ xanthohumol in the near future.…”

Methods for verifying the authenticity of hops - an effective tool against falsification

“…Většina SSR markerů se nachází v nekódujících oblastech genomu. Nárůst informací pomocí sekvenování nové generace (NGS) transkriptomu (Nagel et al, 2008;Clark et al, 2013;Xu et al, 2013) a celého genomu (Natsume et al, 2015) zcela naplnil DNA sekvence genů chmele v EST databázích GeneBank, které tak poskytly možnost vyhledat nové specifi cké molekulární markery. Z těchto informací pak byly odvophenols including, apart from proanthocyanidins, also e.g.…”

“…The majority of SSR markers occur in non-coding regions of the genome. The increasing information by Next Generation Sequencing (NGS) about transcriptome (Nagel et al, 2008;Clark et al, 2013;Xu et al, 2013) and whole genome sequences (Natsume et al, 2015) complemented hop DNA gene sequences in GeneBank EST databases, which have provided the possibility to look for new gene specifi c molecular markers. From this information, a new type of molecular markers was derived such as Expressed Sequence Tag-Simple Sequence Repeat (EST-SSR) (Patzak and Matoušek, 2011;Jakše et al, 2011;Koelling et al, 2012;Singh et al, 2012) and Single Nucleotide Polymorphism (SNP) markers (Matthews et al, 2013;Yamauchi et al, 2014;Henning et al, 2015).…”