Transcriptome analysis of bitter acid biosynthesis and precursor pathways in hop (Humulus lupulus)

Abstract

BackgroundBitter acids (e.g. humulone) are prenylated polyketides synthesized in lupulin glands of the hop plant (Humulus lupulus) which are important contributors to the bitter flavour and stability of beer. Bitter acids are formed from acyl-CoA precursors derived from branched-chain amino acid (BCAA) degradation and C5 prenyl diphosphates from the methyl-D-erythritol 4-phosphate (MEP) pathway. We used RNA sequencing (RNA-seq) to obtain the transcriptomes of isolated lupulin glands, cones with glands removed and leaves from high α-acid hop cultivars, and analyzed these datasets for genes involved in bitter acid biosynthesis including the supply of major precursors. We also measured the levels of BCAAs, acyl-CoA intermediates, and bitter acids in glands, cones and leaves.ResultsTranscripts encoding all the enzymes of BCAA metabolism were significantly more abundant in lupulin glands, indicating that BCAA biosynthesis and subsequent degradation occurs in these specialized cells. Branched-chain acyl-CoAs and bitter acids were present at higher levels in glands compared with leaves and cones. RNA-seq analysis showed the gland-specific expression of the MEP pathway, enzymes of sucrose degradation and several transcription factors that may regulate bitter acid biosynthesis in glands. Two branched-chain aminotransferase (BCAT) enzymes, HlBCAT1 and HlBCAT2, were abundant, with gene expression quantification by RNA-seq and qRT-PCR indicating that HlBCAT1 was specific to glands while HlBCAT2 was present in glands, cones and leaves. Recombinant HlBCAT1 and HlBCAT2 catalyzed forward (biosynthetic) and reverse (catabolic) reactions with similar kinetic parameters. HlBCAT1 is targeted to mitochondria where it likely plays a role in BCAA catabolism. HlBCAT2 is a plastidial enzyme likely involved in BCAA biosynthesis. Phylogenetic analysis of the hop BCATs and those from other plants showed that they group into distinct biosynthetic (plastidial) and catabolic (mitochondrial) clades.ConclusionsOur analysis of the hop transcriptome significantly expands the genomic resources available for this agriculturally-important crop. This study provides evidence for the lupulin gland-specific biosynthesis of BCAAs and prenyl diphosphates to provide precursors for the production of bitter acids. The biosynthetic pathway leading to BCAAs in lupulin glands involves the plastidial enzyme, HlBCAT2. The mitochondrial enzyme HlBCAT1 degrades BCAAs as the first step in the catabolic pathway leading to branched chain-acyl-CoAs.

A feedback insensitive isopropylmalate synthase affects acylsugar composition in cultivated and wild tomato

“…BCAA metabolism has a central role in producing a wide variety of alkyl-acylated plant specialized metabolites (Hirosawa et al, 1995;Pérez et al, 2002;Karppinen et al, 2007;Wang et al, 2008;Clark et al, 2013;Zabalza et al, 2013), making it of interest to understand the various regulatory mechanisms that control flux from amino acid metabolism into specialized metabolite production. We describe the characterization of SlIPMS3, a gene encoding a variant IPMS enzyme that is expressed in the apical cells of type I/IV trichomes in the cultivated tomato S. lycopersicum.…”

Recent transcriptomics advances and emerging applications in food science

“…Recently, Illumina sequencing was also employed for de-novo assembly of hop, turmeric and tea transcriptomes [50][51][52]. In the report on hop, transcriptome data were complemented with metabolite analysis, providing evidence for the lupulin glandspecific branched-chain amino acid and isoprenoid metabolism to produce precursors for bitter acids, which are important compounds in the brewing industry [50].…”

A Heteromeric Membrane-Bound Prenyltransferase Complex from Hop Catalyzes Three Sequential Aromatic Prenylations in the Bitter Acid Pathway

“…The efficiency of prenyltransferase enzymes in hop trichomes is assumed to be very high, as intermediate compounds (e.g. acylphloroglucinol derivatives and naringenin chalcone [NC]) are barely detectable as compared with the very high concentrations of the prenylated products (Nagel et al, 2008;Clark et al, 2013;Xu et al, 2013). In addition to the aforementioned major prenylated chemicals, geranylated chemicals also have been detected in hop tissues, although these occur at lower concentrations (Stevens et al, 2000).…”