A heteromeric membrane-bound prenyltransferase complex from hop catalyzes three sequential aromatic prenylations in the bitter acid pathway

Abstract

Bitter acids (α and β types) account for more than 30% of the fresh weight of hop (Humulus lupulus) glandular trichomes and are well known for their contribution to the bitter taste of beer. These multiprenylated chemicals also show diverse biological activities, some of which have potential benefits to human health. The bitter acid biosynthetic pathway has been investigated extensively, and the genes for the early steps of bitter acid synthesis have been cloned and functionally characterized. However, little is known about the enzyme(s) that catalyze three sequential prenylation steps in the β-bitter acid pathway. Here, we employed a yeast (Saccharomyces cerevisiae) system for the functional identification of aromatic prenyltransferase (PT) genes. Two PT genes (HlPT1L and HlPT2) obtained from a hop trichome-specific complementary DNA library were functionally characterized using this yeast system. Coexpression of codon-optimized PT1L and PT2 in yeast, together with upstream genes, led to the production of bitter acids, but no bitter acids were detected when either of the PT genes was expressed by itself. Stepwise mutation of the aspartate-rich motifs in PT1L and PT2 further revealed the prenylation sequence of these two enzymes in β-bitter acid biosynthesis: PT1L catalyzed only the first prenylation step, and PT2 catalyzed the two subsequent prenylation steps. A metabolon formed through interactions between PT1L and PT2 was demonstrated using a yeast two-hybrid system, reciprocal coimmunoprecipitation, and in vitro biochemical assays. These results provide direct evidence of the involvement of a functional metabolon of membrane-bound prenyltransferases in bitter acid biosynthesis in hop.

Figure 1.

The proposed biosynthetic pathway for bitter acids and XN in hop glandular trichomes. The major prenylation steps in the pathway are highlighted in red, and the possible R group in acylphloroglucinols, DD-acylphloroglucinols, and bitter acids are isobutyryl, isopropyl, and butan-2-yl groups. CCL, Carboxyl CoA ligase; CHS, chalcone synthase; CoASH, CoA; MT, methyltransferase.

Figure 2.

In vivo production of 8DN in various yeast strains. A, Chemical profiling of different yeast strains by liquid chromatography-electrospray ionization-mass spectrometry. For 8DN, retention time = 3.54 min and the MRM transition is mass-to-charge ratio (m/z) 339→119; for naringenin (N), retention time = 1.73 min and the MRM mode is m/z 271→119; for NC, retention time = 1.60 min and the multireaction monitoring (MRM) mode is m/z 271→151. B, Bioconversion ratio of 8DN from the sum of naringenin and NC in different yeast strains. All experiments were performed in triplicate.

Figure 3.

Characterization of HlPT1L and HlPT2 in hop. A, Membrane topology diagram of HlPT1L and HlPT2. The transmembrane domains and the Asp-rich motifs are highlighted in gray and red, respectively. B, Quantitative reverse transcription-PCR analysis of two PT genes in different tissues and different development stages of cones and trichomes. Transcript levels are expressed relative to the Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene (n = 3). WAF, Weeks after flowering. C, Subcellular localizations of HlPT1L (the first 86 amino acids at the N terminus) and HlPT2 (the first 83 amino acids at the N terminus) in Arabidopsis leaf mesophyll protoplasts as revealed by laser confocal microscopy. Chloroplasts are revealed by red chlorophyll autofluorescence. SP, Signal peptide. Bars = 10 μm.

Figure 4.

In vivo production of prenylated chemicals in yeast cells coexpressing HlCCL2, HlCCL4, HlVPS, and different hop aromatic prenyltransferase genes. A, Chromatogram of selected ions of m/z 197.0808 for PIBP, m/z 211.0965 for PIVP, m/z 265.1434 for MD-PIBP, m/z 279.1591 for MD-PIVP, m/z 333.206 for DD-PIBP and G-PIBP, m/z 347.2217 for DD-PIVP and G-PIVP, m/z 401.2715 for colupulone, and m/z 415.2863 for lupulone and adlupulone, all with a mass accuracy in the 20 parts per million range. Some regions of these chromatograms have been rescaled to allow easier visualization. B, Production of different chemicals by yeast strains harboring no PT gene (control), PT1L, PT2, PT1L/PT2, or PT1L-T/PT2-T (the combination of truncated HlPT1L and HlPT2). Data are means ± sd for at least three independent clones. All PT genes used in this experiment were Arabidopsis codon-optimized sequences.

Figure 5.

Effects of the Asp-rich motif on bitter acid production in engineered yeast strains. A, Sequence comparison of wild-type (control) and mutant PT1L/PT2. Only the Asp-rich motifs are shown, and the mutated amino acids are in boldface and italics. B, Production of different compounds by yeast strains harboring wild-type and mutated PT1L/PT2 pairs. Data are means ± sd for at least three independent clones. All PT genes used in this experiment were Arabidopsis codon-optimized sequences.

Figure 6.

Direct interaction between PT1L and PT2 in yeast membranes. A, Split-ubiquitin assays for PT1L and PT2 protein interactions. The different transformed yeast cells were serially diluted and spotted onto synthetic dextrose growth medium (SD, Trp⁻ Leu⁻) and synthetic dextrose selection medium (SD, Trp⁻ Leu⁻ His⁻), both supplemented with 50 mm 3-amino-1,2,4-triazole (3-AT). The positive interaction between PT1L and PT2 was confirmed by filter assay for the detection of 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside acid (X-gal) activity. B, Reciprocal coimmunoprecipitation of His-tagged truncated PT1L and HA-tagged truncated PT2 in yeast cells using anti-HA and anti-His antibodies. Total protein extracts from transformed yeast strains harboring truncated PT1L-At and PT2-At alone or together were incubated with anti-His and anti-HA antibodies, and the immunoprecipitates (IP) were separated by SDS-PAGE and subjected to immunoblot analysis using anti-HA and anti-His antibodies. His-pESC-LEU and HA-pESC-HIS (producing the His-tag or the HA-tag peptide only in yeast cells) were used as negative controls. Ab, Antibody.