Key Enzymes Involved in the Synthesis of Hops Phytochemical Compounds: From Structure, Functions to Applications

Humulus lupulus L. is an essential source of aroma compounds, hop bitter acids, and xanthohumol derivatives mainly exploited as flavourings in beer brewing and with demonstrated potential for the treatment of certain diseases. To acquire a comprehensive understanding of the biosynthesis of these compounds, the primary enzymes involved in the three major pathways of hops’ phytochemical composition are herein critically summarized. Hops’ phytochemical components impart bitterness, aroma, and antioxidant activity to beers. The biosynthesis pathways have been extensively studied and enzymes play essential roles in the processes. Here, we introduced the enzymes involved in the biosynthesis of hop bitter acids, monoterpenes and xanthohumol derivatives, including the branched-chain aminotransferase (BCAT), branched-chain keto-acid dehydrogenase (BCKDH), carboxyl CoA ligase (CCL), valerophenone synthase (VPS), prenyltransferase (PT), 1-deoxyxylulose-5-phosphate synthase (DXS), 4-hydroxy-3-methylbut-2-enyl diphosphate reductase (HDR), Geranyl diphosphate synthase (GPPS), monoterpene synthase enzymes (MTS), cinnamate 4-hydroxylase (C4H), chalcone synthase (CHS_H1), chalcone isomerase (CHI)-like proteins (CHIL), and O-methyltransferase (OMT1). Furthermore, research advancements of each enzyme in terms of reaction conditions, substrate recognition, enzyme structures, and use in engineered microbes are described in depth. Hence, an extensive review of the key enzymes involved in the phytochemical compounds of hops will provide fundamentals for their applications in beer production.

Biosynthesis of pinene in purple non-sulfur photosynthetic bacteria

Background Pinene is a monoterpene, that is used in the manufacture of fragrances, insecticide, fine chemicals, and renewable fuels. Production of pinene by metabolic-engineered microorganisms is a sustainable method. Purple non-sulfur photosynthetic bacteria belong to photosynthetic chassis that are widely used to synthesize natural chemicals. To date, researches on the synthesis of pinene by purple non-sulfur photosynthetic bacteria has not been reported, leaving the potential of purple non-sulfur photosynthetic bacteria synthesizing pinene unexplored. Results Rhodobacter sphaeroides strain was applied as a model and engineered to express the fusion protein of heterologous geranyl diphosphate synthase (GPPS) and pinene synthase (PS), hence achieving pinene production. The reaction condition of pinene production was optimized and 97.51 μg/L of pinene was yielded. Then, genes of 1-deoxy-d-xylulose 5-phosphate synthase, 1-deoxy-d-xylulose 5-phosphate reductoisomerase and isopentenyl diphosphate isomerase were overexpressed, and the ribosome binding site of GPPS-PS mRNA was optimized, improving pinene titer to 539.84 μg/L. Conclusions In this paper, through heterologous expression of GPPS-PS, pinene was successfully produced in R. sphaeroides, and pinene production was greatly improved by optimizing the expression of key enzymes. This is the first report on pinene produce by purple non-sulfur photosynthetic bacteria, which expands the availability of photosynthetic chassis for pinene production.

Biosynthesis of Pinene in Purple Non-Sulfur Photosynthetic Bacteria

Background: Pinene is a monoterpene, that is used in the manufacture of fragrances, insecticide, fine chemicals, and renewable fuels. Production of pinene by metabolic-engineered microorganisms is a sustainable method. Purple non-sulfur photosynthetic bacteria belong to photosynthetic chassis that are widely used to synthesize natural chemicals. To date, researches on the synthesis of pinene by purple non-sulfur photosynthetic bacteria has not been reported, leaving the potential of purple non-sulfur photosynthetic bacteria synthesizing pinene unexplored. Results: Rhodobacter sphaeroides strain was applied as a model and engineered to express the fusion protein of heterologous geranyl diphosphate synthase (GPPS) and pinene synthase (PS), hence achieving pinene production. The reaction condition of pinene production was optimized and 97.51 μg/L of pinene was yielded. Then, genes of 1-deoxy-D-xylulose 5-phosphate synthase, 1-deoxy-D-xylulose 5-phosphate reductoisomerase and isopentenyl diphosphate isomerase were overexpressed, and the ribosome binding site of GPPS-PS mRNA was optimized, improving pinene titer to 539.84 μg/L. Conclusions: In this paper, through heterologous expression of GPPS-PS, pinene was successfully produced in R. sphaeroides, and pinene production was greatly improved by optimizing the expression of key enzymes. This is the first report on pinene produce by purple non-sulfur photosynthetic bacteria, which expands the availability of photosynthetic chassis for pinene production.

Improved cis-Abienol production through increasing precursor supply in Escherichia coli

cis-Abienol, a natural diterpene-diol isolated from balsam fir (Abies balsamea), can be employed as precursors for the semi-synthesis of amber compounds, which are sustainable replacement for ambergris and widely used in the fragmented industry. This study combinatorially co-expressed geranyl diphosphate synthase, geranylgeranyl diphosphate synthase, Labda-13-en-8-ol diphosphate synthase and diterpene synthase, with the best combination achieving ~ 0.3 mg/L of cis-abienol. An additional enhancement of cis-abienol production (up to 8.6 mg/L) was achieved by introducing an exogenous mevalonate pathway which was divided into the upper pathway containing acetyl-CoA acetyltransferase/HMG-CoA reductase and HMG-CoA synthase and the lower pathway containing mevalonate kinase, phosphomevalonate kinase, pyrophosphate mevalonate decarboxylase and isopentenyl pyrophosphate isomerase. The genetically modified strain carrying chromosomal copy of low genes of the mevalonate with the trc promoter accumulated cis-abienol up to 9.2 mg/L in shake flask. Finally, cis-abienol titers of ~ 220 mg/L could be achieved directly from glucose using this de novo cis-abienol-producing E. coli in high-cell-density fermentation. This study demonstrates a microbial process to apply the E. coli cell factory in the biosynthesis of cis-abienol.

Identification and Molecular Characterization of Geranyl Diphosphate Synthase (GPPS) Genes in Wintersweet Flower

Geranyl diphosphate synthase (GPPS) is a plastid localized enzyme that catalyzes the biosynthesis of Geranyl diphosphate (GPP), which is a universal precursor of monoterpenes. Wintersweet (Chimonanthus praecox L.), a famous deciduous flowering shrub with a strong floral scent character, could have GPPS-like homologs that are involved in monoterpenes biosynthesis, but it remains unclear. In the present study, five full-length GPPS and geranylgeranyl diphosphate synthases (GGPPS) genes were identified in the wintersweet transcriptome database. The isolated cDNAs showed high protein sequence similarity with the other plants GPPS and GGPPS. The phylogenetic analysis further classified these cDNAs into four distinct clades, representing heterodimeric GPPS small subunits (SSU1 and SSU2), homodimeric GPPS, and GGPPS. Analysis of temporal expression revealed that all genes have the highest transcript level at the full-open flower stage. From tissue-specific expression analysis, CpGPPS.SSU1 and CpGGPPS1 were predominantly expressed in petal and flower, whereas CpGPPS.SSU2, GPPS, and GGPPS2 showed a constitutive expression. Additionally, the subcellular localization assay identified the chloroplast localization of SSUs and GGPPSs proteins, and the yeast two-hybrid assay showed that both CpGPPS.SSU1 and CpGPPS.SSU2 can interact with the GGPPS proteins. Taken together, these preliminary results suggest that the heterodimeric GPPS can regulate floral scent biosynthesis in wintersweet flower.

Novel routes towards bioplastics from plants: elucidation of the methylperillate biosynthesis pathway from Salvia dorisiana trichomes

Plants produce a large variety of highly functionalized terpenoids. Functional groups such as partially unsaturated rings and carboxyl groups provide handles to use these compounds as feedstock for biobased commodity chemicals. For instance, methylperillate, a monoterpenoid found in Salvia dorisiana, may be used for this purpose, as it carries both an unsaturated ring and a methylated carboxyl group. The biosynthetic pathway of methylperillate in plants is still unclear. In this work, we identified glandular trichomes from S. dorisiana as the location of biosynthesis and storage of methylperillate. mRNA from purified trichomes was used to identify four genes that can encode the pathway from geranyl diphosphate towards methylperillate. This pathway includes a (–)-limonene synthase (SdLS), a limonene 7-hydroxylase (SdL7H, CYP71A76), and a perillyl alcohol dehydrogenase (SdPOHDH). We also identified a terpene acid methyltransferase, perillic acid O-methyltransferase (SdPAOMT), with homology to salicylic acid OMTs. Transient expression in Nicotiana benthamiana of these four genes, in combination with a geranyl diphosphate synthase to boost precursor formation, resulted in production of methylperillate. This demonstrates the potential of these enzymes for metabolic engineering of a feedstock for biobased commodity chemicals.

The Differential Expression of Mevalonate Pathway Genes in the Gut of the Bark Beetle Dendroctonus rhizophagus (Curculionidae: Scolytinae) Is Unrelated to the de Novo Synthesis of Terpenoid Pheromones

Bark beetles commonly produce de novo terpenoid pheromones using precursors synthesized through the mevalonate pathway. This process is regulated by Juvenile Hormone III (JH III). In this work, the expression levels of mevalonate pathway genes were quantified after phloem feeding—to induce the endogenous synthesis of JH III—and after the topical application of a JH III solution. The mevalonate pathway genes from D. rhizophagus were cloned, molecularly characterized, and their expression levels were quantified. Also, the terpenoid compounds produced in the gut were identified and quantified by Gas Chromatography Mass Spectrometry (GC-MS). The feeding treatment produced an evident upregulation, mainly in acetoacetyl-CoA thiolase (AACT), 3-hydroxy-3-methylglutaryl-CoA synthase (HMGS), 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR), phosphomevalonate kinase (PMK), and isopentenyl diphosphate isomerase (IPPI) genes, and males reached higher expression levels compared to females. In contrast, the JH III treatment did not present a clear pattern of upregulation in any sex or time. Notably, the genes responsible for the synthesis of frontalin and ipsdienol precursors (geranyl diphosphate synthase/farnesyl diphosphate synthase (GPPS/FPPS) and geranylgeranyl diphosphate synthase (GGPPS)) were not clearly upregulated, nor were these compounds further identified. Furthermore, trans-verbenol and myrtenol were the most abundant compounds in the gut, which are derived from an α-pinene transformation rather than de novo synthesis. Hence, the expression of mevalonate pathway genes in D. rhizophagus gut is not directed to the production of terpenoid pheromones, regardless of their frequent occurrence in the genus Dendroctonus.

Illustrating and Enhancing the Biosynthesis of Astaxanthin and Docosahexaenoic Acid in Aurantiochytrium sp. SK4

The marine thraustochytrids are a promising source of docosahexaenoic acid (DHA) and the ketocarotenoid astaxanthin. In this study, the biosynthetic pathways of these two important metabolites in Aurantiochytrium sp. SK4 was illustrated by the analyses of the genome, transcriptome, key enzymes, and pathway products. Two sets of genes were involved in two pathways for the biosynthesis of fatty acids. The absence of Δ-15 desaturase genes and the presence of docosapentaenoic acid (DPA), up to 12% of total fatty acids suggest that Aurantiochytrium sp. SK4 may synthesize DHA mainly via a polyketide synthase (PKS) pathway. Three enzymes, namely geranyl diphosphate synthase (GPPS), farnysyl diphosphate synthase (FPPS), and geranylgeranyle diphosphate synthase (GGPPS) were found to be involved in the formation of GGPP that was subsequently catalyzed to β-carotene by a trifunctional CrtIBY enzyme. β-Carotene might be ketolated and then hydroxylated into astaxanthin based on the carotenoid profiles. The formation of GGPP was proposed to be the limiting steps for carotenoid production. Overexpression of the Archaeoglobus GPS together with the Escherichia coli isopentenyl pyrophosphate isomerase, and Vitreoscilla hemoglobin resulted in not only 1.85- and 5.02-fold increases of total carotenoids and astaxanthin, but also 2.40- and 2.74-fold increases of total fatty acids and DHA. This study provides insights into the biosynthesis of carotenoids and fatty acids in Aurantiochytrium.