Biosynthesis Of Bitter Acids Research Articles

Developmental regulation of lupulin gland-associated genes in aromatic and bitter hops (Humulus lupulus L.)

BackgroundHop (Humulus lupulus L.) bitter acids are valuable metabolites for the brewing industry. They are biosynthesized and accumulate in glandular trichomes of the female inflorescence (hop cone). The content of alpha bitter acids, such as humulones, in hop cones can differentiate aromatic from bitter hop cultivars. These contents are subject to genetic and environmental control but significantly correlate with the number and size of glandular trichomes (lupulin glands).ResultsWe evaluated the expression levels of 37 genes involved in bitter acid biosynthesis and morphological and developmental differentiation of glandular trichomes to identify key regulatory factors involved in bitter acid content differences. For bitter acid biosynthesis genes, upregulation of humulone synthase genes, which are important for the biosynthesis of alpha bitter acids in lupulin glands, could explain the higher accumulation of alpha bitter acids in bitter hops. Several transcription factors, including HlETC1, HlMYB61 and HlMYB5 from the MYB family, as well as HlGLABRA2, HlCYCB2–4, HlZFP8 and HlYABBY1, were also more highly expressed in the bitter hop cultivars; therefore, these factors may be important for the higher density of lupulin glands also seen in the bitter hop cultivars.ConclusionsGene expression analyses enabled us to investigate the differences between aromatic and bitter hops. This study confirmed that the bitter acid content in glandular trichomes (lupulin glands) is dependent on the last step of alpha bitter acid biosynthesis and glandular trichome density.

Gene Expression of Agronomically Important Secondary Metabolites in cv. ‘USDA Cascade’ Hop (Humulus lupulus L.) Cones during Critical Developmental Stages

The transcriptome from lupulin glands and associated bracts from cone tissue of hop (Humulus lupulus) c.v. ‘Cascade’, during three stages of development: early, mid, and late or near-harvest, was sequenced. Significant increases were found in expression patterns of many genes involved in the biosynthesis of bitter acids, xanthohumol, and volatile secondary metabolites or “hop oils” during the middle stage of cone development. The biosynthesis of thiol precursors responsible for popular “tropical fruit” flavors in beer is not well known, but homologs of genes hypothesized to be involved in this process tend to be up-regulated during the late stage in hop cones. More research needs to be performed to describe the pathway of thiol precursor biosynthesis in hops. Hierarchical clustering revealed overlap of samples taken from each developmental stage, likely due to non-uniform ripening of cones on the plant. It is proposed that the mid-stage of cone development is critical for the development of important flavor-producing secondary metabolites in hops, and this is supported by previous research describing concentrations of secondary metabolites. It is hypothesized that abiotic stress during the mid-stage of cone development may be quite detrimental to the bitter acid concentrations ultimately found in hops.

Agronomic and chemical evaluation of hop cultivars grown under Mediterranean conditions

Aim of study: Evaluation of the agronomic performance and chemical profile of four hop cultivars grown under Mediterranean conditions.Area of study: The study was undertaken in Bragança, north-eastern Portugal.Material and methods: The newly introduced cultivars (‘Columbus’, ‘Cascade’ and ‘Comet’) were compared with the well-stablished ‘Nugget’. The field experiment was carried out between 2017 and 2019. Dry matter (DM) yield (plant and cones), tissue elemental composition and bitter acid and nitrate (NO3-) concentrations in the cones were assessed.Main results: ‘Comet’ was the most productive cultivar with the highest total DM yield (1,624 to 1,634 g plant-1), cone yield (572 to 633 g plant-1), and dry weight of individual cones (0.28 to 0.79 g cone-1). ‘Cascade’ showed the lowest average total DM yield (723 to 1,045 g plant-1). The year affected the average values of DM yield and the concentration of bitter acids in the cones, with ‘Cascade’ showing the highest sensitivity between cultivars. The concentrations of α and β-acids in the cones were within or close to the normal ranges internationally accepted for all cultivars. ‘Columbus’ exhibited the highest levels of α-acids, ranging between 12.04 % and 12.23%, followed by ‘Nugget’ (10.17–11.90%), ‘Comet’ (9.32–10.69%) and ‘Cascade’ (4.46–8.72%). The nutrient accumulation criteria in cone and leaf tissues seem to be a differentiating factor between cultivars with influence on bitter acid biosynthesis and biomass production.Research highlights: All cultivars showed notable performance in terms of DM yield and bitter acid concentration in the cones when compared to international standards.

Hop dry matter yield and cone quality responses to amino acid and potassium-rich foliar spray applications

The use of amino acid and K-rich foliar sprays was evaluated in a commercial hop field in North-eastern Portugal. Four applications of an amino acid-rich foliar spray were performed in place of a second side dress N application of ⁓70 kg N ha−1, which is usually applied by the farmer. The K-rich foliar spray was applied once at the cone developing stage as a supplement to the farmer’s fertilization plan. The amino acid-enriched foliar spray maintained crop dry matter yield at the levels of the control treatment and increased cone alpha-acids concentration (41.8% in 2018 and 9.3% in 2019). Foliar K did not increase cone dry matter yield, cone size or bitter acid concentration. Tissue K concentration was not significantly affected by foliar treatments whereas the application of K seemed to increase N uptake, with leaves and stems being the predominant allocation tissues. Both foliar treatments increased leaf and stem Mg concentrations. The results seem to emphasize the importance of amino acids in the biosynthesis of bitter acids, while K and Zn seemed to play an important secondary role, maybe related to N metabolism and its reduction into amino acids. The concentrations of total phenols in cones and leaves were lower in the foliar treatments in comparison to the control, and the higher values registered in leaves. In this study, the use of amino acids as a foliar spray provided an interesting result, since they maintained cone dry matter yield and increased cone bitter acid concentration with reduced N use.

Genome-wide transcriptome profiling of transgenic hop (Humulus lupulus L.) constitutively overexpressing HlWRKY1 and HlWDR1 transcription factors

BackgroundThe hop plant (Humulus lupulus L.) is a valuable source of several secondary metabolites, such as flavonoids, bitter acids, and essential oils. These compounds are widely implicated in the beer brewing industry and are having potential biomedical applications. Several independent breeding programs around the world have been initiated to develop new cultivars with enriched lupulin and secondary metabolite contents but met with limited success due to several constraints. In the present work, a pioneering attempt has been made to overexpress master regulator binary transcription factor complex formed by HlWRKY1 and HlWDR1 using a plant expression vector to enhance the level of prenylflavonoid and bitter acid content in the hop. Subsequently, we performed transcriptional profiling using high-throughput RNA-Seq technology in leaves of resultant transformants and wild-type hop to gain in-depth information about the genome-wide functional changes induced by HlWRKY1 and HlWDR1 overexpression.ResultsThe transgenic WW-lines exhibited an elevated expression of structural and regulatory genes involved in prenylflavonoid and bitter acid biosynthesis pathways. In addition, the comparative transcriptome analysis revealed a total of 522 transcripts involved in 30 pathways, including lipids and amino acids biosynthesis, primary carbon metabolism, phytohormone signaling and stress responses were differentially expressed in WW-transformants. It was apparent from the whole transcriptome sequencing that modulation of primary carbon metabolism and other pathways by HlWRKY1 and HlWDR1 overexpression resulted in enhanced substrate flux towards secondary metabolites pathway. The detailed analyses suggested that none of the pathways or genes, which have a detrimental effect on physiology, growth and development processes, were induced on a genome-wide scale in WW-transgenic lines.ConclusionsTaken together, our results suggest that HlWRKY1 and HlWDR1 simultaneous overexpression positively regulates the prenylflavonoid and bitter acid biosynthesis pathways in the hop and thus these transgenes are presented as prospective candidates for achieving enhanced secondary metabolite content in the hop.

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

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.

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

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.

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

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.

HlPT-1, a membrane-bound prenyltransferase responsible for the biosynthesis of bitter acids in hops

Female flowers of hop (Humulus lupulus L.) develop a large number of glandular trichomes called lupulin glands that contain a variety of prenylated compounds such as α- and β-acid (humulone and lupulone, respectively), as well as xanthohumol, a chalcone derivative. These prenylated compounds are biosynthesized by prenyltransferases catalyzing the transfer of dimethylallyl moiety to aromatic substances. In our previous work, we found HlPT-1 a candidate gene for such a prenyltransferase in a cDNA library constructed from lupulin-enriched flower tissues. In this study, we have characterized the enzymatic properties of HlPT-1 using a recombinant protein expressed in baculovirus-infected insect cells. HlPT-1 catalyzed the first transfer of dimethylallyl moiety to phloroglucinol derivatives, phlorisovalerophenone, phlorisobutyrophenone and phlormethylbutanophenone, leading to the formation of humulone and lupulone derivatives. HlPT-1 also recognized naringenin chalcone as a flavonoid substrate to yield xanthohumol, and this broad substrate specificity is a unique character of HlPT-1 that is not seen in other reported flavonoid prenyltransferases, all of which show strict specificity for their aromatic substrates. Moreover, unlike other aromatic substrate prenyltransferases, HlPT-1 revealed an exclusive requirement for Mg2+ as a divalent cation for its enzymatic activity and also showed exceptionally narrow optimum pH at around pH 7.0.

DNA Sequence and Expression Variation of Hop (Humulus lupulus) Valerophenone Synthase (VPS), a Key Gene in Bitter Acid Biosynthesis

The hop plant (Humulus lupulus) is a source of many secondary metabolites, with bitter acids essential in the beer brewing industry and others having potential applications for human health. This study investigated variation in DNA sequence and gene expression of valerophenone synthase (VPS), a key gene in the bitter acid biosynthesis pathway of hop. Sequence variation was studied in 12 varieties, and expression was analysed in four of the 12 varieties in a series across the development of the hop cone. Nine single nucleotide polymorphisms (SNPs) were detected in VPS, seven of which were synonymous. The two non-synonymous polymorphisms did not appear to be related to typical bitter acid profiles of the varieties studied. However, real-time quantitative reverse-transcription polymerase chain reaction (qRT-PCR) analysis of VPS expression during hop cone development showed a clear link with the bitter acid content. The highest levels of VPS expression were observed in two triploid varieties, 'Symphony' and 'Ember', which typically have high bitter acid levels. In all hop varieties studied, VPS expression was lowest in the leaves and an increase in expression was consistently observed during the early stages of cone development.

Valerophenone synthase-like chalcone synthase homologues in Humulus lupulus

Valerophenone synthase homologue of chalcone synthase (CHS) is the first key enzyme described to be involved in the biosynthesis of bitter acids, the compounds produced in hop lupulin glands valuable for the taste of beer. The complete sequence of a novel homologue of CHS chs 4 was isolated from hop. Protein predicted from chs 4 cDNA has 43.45 kDa and length 395 amino acids. It was found by the analysis of chs 4 flanking sequences that this gene is in the cluster with other CHS homologues — chs 3 and vps. The intron identified in chs 4 has been found to be homological to vps and chs 3 introns. Expression of chs 4 was partially characterized using reverse transcription polymerase chain reaction and it was found that chs 4 is specifically expressed in the glandular tissue of hop cones likewise vps. The predicted protein sequence CHS 4 was compared with other CHS-like proteins.

Analysis of the chalcone synthase from Humulus lupulus L. and biotechnology aspects of medicinal hops

The complete sequence hop gene, which corresponds to true chalcone synthase (EC 2.3.1.74), was amplified using a combination of PCR, RT PCR and inverse PCR methods and cloned from Czech Osvald’s clone 72. The gene designated chs_H1 was found to be specifically expressed on glandular trichomes, whereas negligible level of specific mRNA was found in leaves. Thus, chs_H1 may co-determine biosynthesis of prenylated chalcones, compounds valuable as anticancer and antiproliferative components of lupulin. It was shown by the comparative analyses and by the structure modelling that the true hop chalcone synthase differs from previously described CHS-like protein, phlorisovalerophenone synthase, which is involved in biosynthesis of bitter acids. Several hop cultivars were analysed for the presence of genes homologous to chs_H1 using chs_H1 cDNA as probe. 2-4 HindIII specific genomic fragments were detected by Southern blots, depending on cultivar. More detailed analysis revealed at least six homologous genes in Osvald’s 72 hop, suggesting a great potential of this hop also as a genetic source for modern biotechnology. High level of xanthohumol in some Osvald’s 72-derived hybrid cultivars like cv. Sládek, as well as a comparatively high level of prenylated flavonoids in some comprehensive hops, suggest also the presence of additional, regulatory genes co-determining levels of prenylated flavonoids valuable for medicinal hops.

Biosynthesis of bitter acids in hops. A (13)C-NMR and (2)H-NMR study on the building blocks of humulone.

The biosynthesis of humulone, an antibacterial bitter acid from hops, was studied by isotope-incorporation experiments using (13)C-labelled glucose or (2)H(2)O. (13)C enrichments, (2)H enrichments and (13)C(13)C coupling patterns identify isovaleryl-CoA, malonyl-CoA and dimethylallyl pyrophosphate as precursors for humulone. Dimethylallyl pyrophosphate, which serves as a building block for the bitter acid, is generated via the deoxyxylulose pathway of terpenoid biosynthesis. The data confirm that a symmetrical intermediate is involved in humulone formation.

ChemInform Abstract: In vitro Prenylation of Aromatic Intermediates in the Biosynthesis of Bitter Acids in Humulus lupulus.

AbstractChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 100 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a “Full Text” option. The original article is trackable via the “References” option.

In-vitro prenylation of aromatic intermediates in the biosynthesis of bitter acids in humulus lupulus

Abstract Bitter acids are accumulated in the glandular hairs of hop cones. The first step of the biosynthesis of the bitter acids, namely formation of the aromatic intermediates, is catalysed by a chalcone synthase-like enzyme. In the present paper two sequential prenylation steps, leading to first the production of compounds X (4-prenylphlorisovalerophenone) or co-X (4-prenylphlorisobutyrophenone) and thereafter to the production of deoxyhumulone or deoxycohumulone, respectively, are described. An HPLC assay for these prenylation reactions has been set up, and enzyme activities were measured in protein extracts from hop flowerbuds, flowers and young cones. Furthermore, preliminary characterization experiments of the prenyltransferases were performed.

Formation of aromatic intermediates in the biosynthesis of bitter acids in Humulus lupulus

A new hypothesis concerning the formation of aromatic intermediates in the biosynthesis of hop bitter acids was tested. Two phloroglucinol derivatives, 2-(3-methylbutanoyl)-1,3,5-benzenetriol (phlorisovalerophenone = PIVP) and 2-(2-methylpropanoyl)-1,3,5-benzenetriol (phlorisobutyrophenone = PIBP), were detected in hop extracts. The formation of these compounds was shown to occur in vitro in hop protein extracts using the precursors malonyl-CoA plus isovaleryl-CoA or isobutyryl-CoA, respectively. We conclude that PIVP and PIBP are probably the first aromatic intermediates formed in the biosynthesis of humulone/lupulone and cohumulone/colupulone, respectively. We suggest that the reactions are catalysed by a chalcone synthase-like enzyme. The enzyme chalcone synthase and its activity were found to be present in hop protein extracts. The formation of naringenin, PIVP and PIBP in various hop protein extracts obtained during the development from flower bud to ripe cone was compared.

Possible Involvement of Chalcone Synthase in the Biosynthesis of Bitter Acids inHumulus lupulus

Possible Involvement of Chalcone Synthase in the Biosynthesis of Bitter Acids in<i>Humulus lupulus</i>