Pterostilbene production by microorganisms expressing resveratrol O-methyltransferase

Pterostilbene (3,5-dimethoxy-4′-hydroxyl-trans-stilbene)—a derivative of resveratrol—is a natural dietary compound and the primary antioxidant component in berries. Pterostilbene has significant advantages over resveratrol in bioavailability, half-life in the body, cellular uptake, oral absorption and metabolic stability. Here, we expressed the resveratrol O-methyltransferase (ROMT) gene (VvROMT) from grape (Vitis vinifera) in Escherichia coli and Saccharomyces cerevisiae and confirmed its specific ability to catalyze the production of pterostilbene from resveratrol. By co-expressing an additional two genes from the resveratrol biosynthetic pathway—4-coumarate CoA-ligase (4CL) and stilbene synthase (STS)—a large amount of pterostilbene was produced, with a trace amount of pinostilbene detected. To understand the molecular basis of the catalytic activity, four key amino acid residues were identified in a 3D-model of VvROMT and mutagenized and assayed for augmented catalytic activity. Our results demonstrate the potential utility of the engineered microorganisms for pterostilbene production and provide protein engineering targets that will hopefully lead to increased activity of the ROMT enzyme.     

Intermediate-sensor assisted push–pull strategy and its application in heterologous deoxyviolacein production in Escherichia coli

Because high-throughput screening tools are typically unavailable when using the pathway-engineering approach, we developed a new strategy, named intermediate sensor-assisted push–pull strategy, which enables sequential pathway optimization by incorporating a biosensor targeting a key pathway intermediate. As proof of concept, we constructed an l-Trp biosensor and used it to optimize the deoxyviolacein biosynthetic pathway, which we divided into two modules with l-Trp being the product of the upstream and the substrate of the downstream module for deoxyviolacein synthesis. Using the biosensor and fluorescence-activated cell sorting, the activities of the two modules were sequentially and independently optimized in Escherichia coli to achieve the desired phenotypes. By this means, we increased the deoxyviolacein titer 4.4-fold (1.92 g/L), which represents the greatest deoxyviolacein production reported. This work suggests that a biosynthetic pathway can be enhanced to produce a value-added secondary metabolite(s) without available end-product screening method by using a central metabolic junction molecule biosensor(s).     

Precision breeding for RNAi suppression of a major 4-coumarate:coenzyme A ligase gene improves cell wall saccharification from field grown sugarcane

Sugarcane (Saccharum spp. hybrids) is a major feedstock for commercial bioethanol production. The recent integration of conversion technologies that utilize lignocellulosic sugarcane residues as well as sucrose from stem internodes has elevated bioethanol yields. RNAi suppression of lignin biosynthetic enzymes is a successful strategy to improve the saccharification of lignocellulosic biomass. 4-coumarate:coenzyme A ligase (4CL) is a key enzyme in the biosynthesis of phenylpropanoid metabolites, such as lignin and flavonoids. Identifying a major 4CL involved in lignin biosynthesis among multiple isoforms with functional divergence is key to manipulate lignin biosynthesis. In this study, two full length 4CL genes (Sh4CL1 and Sh4CL2) were isolated and characterized in sugarcane. Phylogenetic, expression and RNA interference (RNAi) analysis confirmed that Sh4CL1 is a major lignin biosynthetic gene. An intragenic precision breeding strategy may facilitate the regulatory approval of the genetically improved events and was used for RNAi suppression of Sh4CL1. Both, the RNAi inducing cassette and the expression cassette for the mutated ALS selection marker consisted entirely of DNA sequences from sugarcane or the sexually compatible species Sorghum bicolor. Field grown sugarcane with intragenic RNAi suppression of Sh4CL1 resulted in reduction of the total lignin content by up to 16.5?% along with altered monolignol ratios without reduction in biomass yield. Mature, field grown, intragenic sugarcane events displayed 52–76?% improved saccharification efficiency of lignocellulosic biomass compared to wild type (WT) controls. This demonstrates for the first time that an intragenic approach can add significant value to lignocellulosic feedstocks for biofuel and biochemical production.     

In Situ Localization of Phenylpropanoid Biosynthetic mRNAs and Proteins in Parsley (Petroselinum crispum)

Using in situ RNA/RNA hybridization, enzyme immunolocalization, and histochemical techniques, several phenylpropanoid biosynthetic activities and products were localized in tissue sections from various aerial parts of parsley (Petroselinum crispum) plants at different developmental stages. The enzymes and corresponding mRNAs analyzed included two representatives of general phenylpropanoid metabolism: phenylalanine ammonia-lyase (PAL) and 4-coumarate: CoA ligase (4CL), and one representative each from two distinct branch pathways: chalcone synthase (CHS; flavonoids) and S-adenosyl-L-methionine: bergaptol O-methyltransferase (BMT; furanocoumarins). In almost all cases, the relative timing of accumulation differed greatly for mRNA and protein and indicated short expression periods and short half-lives for all mRNAs as compared to the proteins. PAL and 4CL occurred almost ubiquitously in cell type-specific patterns, and their mRNAs and proteins were always coordinately expressed, whereas the cell type-specific localization of flavonoid and furanocoumarin biosynthetic activities was to a large extent mutually exclusive. However, the distribution patterns of CHS and BMT, when superimposed, closely matched those of PAL and 4CL in nearly all tissues analysed, suggesting that the flavonoid and furanocoumarin pathways together consituted a large majority of the total phenylpropanoid biosynthetic activity. Differential sites of synthesis and accumulation indicating intercellular translocation were observed both for flavonoids and for furanocoumarins in oil ducts and the surrounding tissue. The widespread occurrence of both classes of compounds, as well as selected, pathway-specific mRNAs and enzymes, in many cell types of all parsley organs including various flower parts suggests additional functions beyond the previously established roles of flavonoids in UV protection and furanocoumarins in pathogen defence.     

Isopentenyl diphosphate (IPP)-bypass mevalonate pathways for isopentenol production

Branched C₅ alcohols are promising biofuels with favorable combustion properties. A mevalonate (MVA)-based isoprenoid biosynthetic pathway for C₅ alcohols was constructed in Escherichia coli using genes from several organisms, and the pathway was optimized to achieve over 50% theoretical yield. Although the MVA pathway is energetically less efficient than the native methylerythritol 4-phosphate (MEP) pathway, implementing the MVA pathway in bacterial hosts such as E. coli is advantageous due to its lack of endogenous regulation. The MVA and MEP pathways intersect at isopentenyl diphosphate (IPP), the direct precursor to isoprenoid-derived C₅ alcohols and initial precursor to longer chain terpenes, which makes independent regulation of the pathways difficult. In pursuit of the complete “decoupling” of the MVA pathway from native cellular regulation, we designed novel IPP-bypass MVA pathways for C₅ alcohol production by utilizing promiscuous activities of two enzymes, phosphomevalonate decarboxylase (PMD) and an E. coli-endogenous phosphatase (AphA). These bypass pathways have reduced energetic requirements, are further decoupled from intrinsic regulation, and are free from IPP-related toxicity. In addition to these benefits, we demonstrate that reduced aeration rate has less impact on the bypass pathway than the original MVA pathway. Finally, we showed that performance of the bypass pathway was primarily determined by the activity of PMD. We designed PMD mutants with improved activity and demonstrated titer increases in the mutant strains. These modified pathways would be a good platform for industrial production of isopentenol and related chemicals such as isoprene.     

Design and application of an in vivo reporter assay for phenylalanine ammonia-lyase

Phenylalanine ammonia-lyase (PAL) is an important enzyme that links primary metabolism to secondary metabolism. Its efficiency is often a critical factor that affects the overall flux of a related metabolic pathway, the titer of the final products, and the efficacy of PAL-based therapies. Thus, PAL is a common target for metabolic engineering, and it is of significant interest to screen efficient PALs for industrial and medical applications. In this study, a novel and efficient visible reporter assay for screening of PAL efficiency in Escherichia coli was established based on a plant type III polyketide biosynthetic pathway. The candidate PALs were co-expressed with a 4-coumarate:CoA ligase 4CL1 from Arabidopsis thaliana and curcuminoid synthase (CUS) from Oryza sativa in E. coli BL21(DE3) to form a dicinnamoylmethane biosynthetic pathway. Taking advantage of the yellow color of the product, a microplate-based assay was designed to measure the titer of dicinnamoylmethane, which was validated by HPLC analysis. The different titers of the product reflect the overall performance (expression level and enzymatic activity) of the individual PALs in E. coli. Using this system, we have screened three PALs (PAL1, PAL3, and PAL4) from Trifolium pratense, among which PAL1 showed the best performance in E. coli. The engineered E. coli strain containing PAL1, 4CL1, and CUS led to the production of dicinnamoylmethane at a high level of 0.36 g/l. Supplement of 2-fluoro-phenylalanine yielded two fluorinated dicinnamoylmethane derivatives, 6,6′-difluoro-dicinnamoylmethane and 6-fluoro-dicinnamoylmethane, of which the latter is a new curcuminoid.     

Three 4-coumarate:coenzyme A ligases in Arabidopsis thaliana represent two evolutionarily divergent classes in angiosperms

The enzyme 4-coumarate:CoA ligase (4CL) plays a key role in channelling carbon flow into diverse branch pathways of phenylpropanoid metabolism which serve important functions in plant growth and adaptation to environmental perturbations. Here we report on the cloning of the 4CL gene family from Arabidopsis thaliana and demonstrate that its three members, At4CL1, At4CL2 and At4CL3, encode isozymes with distinct substrate preference and specificities. Expression studies revealed a differential behaviour of the three genes in various plant organs and upon external stimuli such as wounding and UV irradiation or upon challenge with the fungus, Peronospora parasitica. Phylogenetic comparisons indicate that, in angiosperms, 4CL can be classified into two major clusters, class I and class II, with the At4CL1 and At4CL2 isoforms belonging to class I and At4CL3 to class II. Based on their enzymatic properties, expression characteristics and evolutionary relationships, At4CL3 is likely to participate in the biosynthetic pathway leading to flavonoids whereas At4CL1 and At4CL2 are probably involved in lignin formation and in the production of additional phenolic compounds other than flavonoids.     

Discovery of the selective sphingomyelin synthase 2 inhibitors with the novel structure of oxazolopyridine

Sphingomyelin synthase (SMS) is a key enzyme in sphingomyelin biosynthetic pathway, whose activity is highly related to the atherosclerosis progression. SMS2 could serve as a promising therapeutic target for atherosclerosis. Based on the structure of lead compound D2, a series of oxazolopyridine derivatives were designed, synthesized, and their inhibitory activities against purified SMS1 and SMS2 enzymes were evaluated respectively. The representative molecules QY4 and QY16 possess micromolar inhibitory activities against SMS2 and excellent isoform preferences over SMS1, qualified to be selected as potential molecules in further discovery of specific SMS2 inhibitors.     

大肠杆菌中从头合成白藜芦醇途径的设计及优化

白藜芦醇是一种极具药用价值的植物源芪类化合物。为了在E. coli实现白藜芦醇的从头合成,构建了由酪氨酸解氨酶（TAL）,香豆酸-CoA合成酶（4CL）和白藜芦醇合成酶（STS）组成的非天然合成途径。经3天发酵后,白藜芦醇产量仅为2.67 mg/L,而其中间体香豆酸的积累达到了95.64 mg/L。为了进一步改善异源途径的效率,对4CL和STS模块采取融合表达、高拷贝表达及启动子工程改造的策略,最终使白藜芦醇产量提高到了9.6倍,达到了25.76 mg/L,同时香豆酸的积累减少到了20.38 mg/L。这些研究结果为更高效白藜芦醇从头合成工程菌的构建及最终实现白藜芦醇的微生物大规模生产奠定了基础。     

Precursor-Directed Combinatorial Biosynthesis of Cinnamoyl,Dihydrocinnamoyl, and Benzoyl Anthranilates in Saccharomyces cerevisiae

Biological synthesis of pharmaceuticals and biochemicals offers an environmentally friendly alternative to conventional chemical synthesis. These alternative methods require the design of metabolic pathways and the identification of enzymes exhibiting adequate activities. Cinnamoyl, dihydrocinnamoyl, and benzoyl anthranilates are natural metabolites which possess beneficial activities for human health, and the search is expanding for novel derivatives that might have enhanced biological activity. For example, biosynthesis in Dianthus caryophyllus is catalyzed by hydroxycinnamoyl/benzoyl-CoA:anthranilate N-hydroxycinnamoyl/ benzoyltransferase (HCBT), which couples hydroxycinnamoyl-CoAs and benzoyl-CoAs to anthranilate. We recently demonstrated the potential of using yeast (Saccharomyces cerevisiae) for the biological production of a few cinnamoyl anthranilates by heterologous co-expression of 4-coumaroyl:CoA ligase from Arabidopsis thaliana (4CL5) and HCBT. Here we report that, by exploiting the substrate flexibility of both 4CL5 and HCBT, we achieved rapid biosynthesis of more than 160 cinnamoyl, dihydrocinnamoyl, and benzoyl anthranilates in yeast upon feeding with both natural and non-natural cinnamates, dihydrocinnamates, benzoates, and anthranilates. Our results demonstrate the use of enzyme promiscuity in biological synthesis to achieve high chemical diversity within a defined class of molecules. This work also points to the potential for the combinatorial biosynthesis of diverse and valuable cinnamoylated, dihydrocinnamoylated, and benzoylated products by using the versatile biological enzyme 4CL5 along with characterized cinnamoyl-CoA- and benzoyl-CoA-utilizing transferases.     

Efficient de novo synthesis of resveratrol by metabolically engineered <Emphasis Type="Italic">Escherichia coli</Emphasis>

Resveratrol has been the subject of numerous scientific investigations due to its health-promoting activities against a variety of diseases. However, developing feasible and efficient microbial processes remains challenging owing to the requirement of supplementing expensive phenylpropanoic precursors. Here, various metabolic engineering strategies were developed for efficient de novo biosynthesis of resveratrol. A recombinant malonate assimilation pathway from Rhizobium trifolii was introduced to increase the supply of the key precursor malonyl-CoA and simultaneously, the clustered regularly interspaced short palindromic repeats interference system was explored to down-regulate fatty acid biosynthesis pathway to inactivate the malonyl-CoA consumption pathway. Down-regulation of fabD, fabH, fabB, fabF, fabI increased resveratrol production by 80.2, 195.6, 170.3, 216.5 and 123.7%, respectively. Furthermore, the combined effect of these genetic perturbations was investigated, which increased the resveratrol titer to 188.1 mg/L. Moreover, the efficiency of this synthetic pathway was improved by optimizing the expression level of the rate-limiting enzyme TAL based on reducing mRNA structure of 5′ region. This further increased the final resveratrol titer to 304.5 mg/L. The study described here paves the way to the development of a simple and economical process for microbial production of resveratrol.     

Characterization and directed evolution of propionyl-CoA carboxylase and its application in succinate biosynthetic pathway with two CO2 fixation reactions

Propionyl-CoA carboxylase (PCC) is a promising enzyme in the fields of biological CO₂ utilization, synthesis of natrual products, and so on. The activity and substrate specificity of PCC are dependent on its key subunit carboxyltransferase (CT). To obtain PCC with high enzyme activity, seven pccB genes encoding CT subunit from diverse microorganisms were expressed in recombinant E. coli, and PccB from Bacillus subtilis showed the highest activity in vitro. To further optimize this protein using directed evolution, a genetic screening system based on oxaloacetate availability was designed to enrich the active variants of PccB_Bs. Four amino acid substitutions (D46G, L97Q, N220I and I391T) proved of great assistance in PccB_Bs activity improvement, and a double mutant of PccB_Bs (N220I/I391T) showed a 94-fold increase of overall catalytic efficiency indicated by k_cat/K_m. Moreover, this PccB_Bs double mutant was applied in construction of new succinate biosynthetic pathway. This new pathway produces succinate from acetyl-CoA with fixation of two CO₂ molecules, which was confirmed by isotope labeling experiment with NaH¹³CO₃. Compared with previous succinate production based on carboxylation of phosphoenolpyruvate or pyruvate, this new pathway showed some advantages including higher CO₂ fixation potentiality and availability under aerobic conditions. In summary, this study developed a PCC with high enzyme activity which can be widely used in biotechnology field, and also demonstrated the feasibility of new succinate biosynthetic pathway with two CO₂ fixation reactions.     

Substrates and enzyme activities related to biotransformation of resveratrol from phenylalanine by Alternaria sp. MG1

To identify the substrates and enzymes related to resveratrol biosynthesis in Alternaria sp. MG1, different substrates were used to produce resveratrol, and their influence on resveratrol production was analyzed using high performance liquid chromatography (HPLC). Formation of resveratrol and related intermediates was identified using mass spectrum. During the biotransformation, activities of related enzymes, including phenylalanine ammonia-lyase (PAL), trans-cinnamate 4-hydroxylase (C4H), and 4-coumarate-CoA ligase (4CL), were analyzed and tracked. The reaction system contained 100 mL 0.2 mol/L phosphate buffer (pH 6.5), 120 g/L Alternaria sp. MG1 cells, 0.1 g/L MgSO₄, and 0.2 g/L CaSO₄ and different substrates according to the experimental design. The biotransformation was carried out for 21 h at 28 °C and 120 rpm. Resveratrol formation was identified when phenylalanine, tyrosine, cinnamic acid, and p-coumaric acid were separately used as the only substrate. Accumulation of cinnamic acid, p-coumaric acid, and resveratrol and the activities of PAL, C4H, and 4CL were identified and changed in different trends during transformation with phenylalanine as the only substrate. The addition of carbohydrates and the increase of phenylalanine concentration promoted resveratrol production and yielded the highest value (4.57 μg/L) when 2 g/L glucose, 1 g/L cyclodextrin, and phenylalanine (4.7 mmol/L) were used simultaneously.     

Improved virus-induced gene silencing allows discovery of a serpentine synthase gene in Catharanthus roseus

Specialized metabolites are chemically complex small molecules with a myriad of biological functions. To investigate plant-specialized metabolite biosynthesis more effectively, we developed an improved method for virus-induced gene silencing (VIGS). We designed a plasmid that incorporates fragments of both the target gene and knockdown marker gene (phytoene desaturase, PDS), which identifies tissues that have been successfully silenced in planta. To demonstrate the utility of this method, we used the terpenoid indole alkaloid (TIA) pathway in Madagascar periwinkle (Catharanthus roseus) as a model system. Catharanthus roseus is a medicinal plant well known for producing many bioactive compounds, such as vinblastine and vincristine. Our VIGS method enabled the discovery of a previously unknown biosynthetic enzyme, serpentine synthase (SS). This enzyme is a cytochrome P450 (CYP) that produces the β-carboline alkaloids serpentine and alstonine, compounds with strong blue autofluorescence and potential pharmacological activity. The discovery of this enzyme highlights the complexity of TIA biosynthesis and demonstrates the utility of this improved VIGS method for discovering unidentified metabolic enzymes in plants.

An improved virus-induced gene silencing approach led to the discovery of the alkaloid biosynthetic enzyme serpentine synthase.     

Action of ultraviolet-C on stilbene formation in callus ofArachis hypogaea

The action of light on the formation of stilbenes and the induction of stilbene synthase in dark-grown and light-grown callus of peanut (Arachis hypogaea) was investigated over the wavelength range from 250 to 400 nm. Ultraviolet light of 260–270 nm had a significant and selective effect on the formation of resveratrol and isopentenylresveratrol. The callus responded by the production of stilbene synthase, with maximal activity appearing 4 h after irradiation with a fluence rate of 1 W m^-2 (270 nm) applied for 10 min. At lower fluence rates, maximal responses in enzyme activity were shifted to longer induction periods. The efficiency of the biosynthetic pathway, and the form and maxima of enzyme profiles depended on the duration of exposure. We failed to demonstrate any significant influence of red light at low energy irradiation (672 nm, 726 nm and 753 nm).     

<Emphasis Type="Italic">Escherichia coli</Emphasis> modular coculture system for resveratrol glucosides production

In bio-based fermentation, the overall bioprocess efficiency is significantly affected by the metabolic burden associated with the expression of complete biosynthetic pathway as well as precursor and cofactor generating enzymes into a single microbial cell. To attenuate such burden by compartmentalizing the enzyme expression, recently synthetic biologists have used coculture or poly-culture techniques for biomolecules synthesis. In this paper, coculture system of two metabolically engineered Escherichia coli populations were employed which comprises upstream module expressing two enzymes converting para-coumaric acid into resveratrol and the downstream module expressing glucosyltransferase to convert the resveratrol into its glucosidated forms; polydatin and resveratroloside. Upon optimization of the initial inoculum ratio of two E. coli populations, 92 mg resveratrol glucosides/L (236 µM) was produced i.e. achieving 84% bioconversion from 280 µM of p-coumaric acid in 60 h by 3 L fed batch fermentor. This is the report of applying coculture system to produce resveratrol glucosides by expressing the aglycone formation pathway and sugar dependent pathway into two different cells.     

Development of bifunctional biosensors for sensing and dynamic control of glycolysis flux in metabolic engineering

Glycolysis is the primary metabolic pathway in all living organisms. Maintaining the balance of glycolysis flux and biosynthetic pathways is the crucial matter involved in the microbial cell factory. Few regulation systems can address the issue of metabolic flux imbalance in glycolysis. Here, we designed and constructed a bifunctional glycolysis flux biosensor that can dynamically regulate glycolysis flux for overproduction of desired biochemicals. A series of positive-and negative-response biosensors were created and modified for varied thresholds and dynamic ranges. These engineered glycolysis flux biosensors were verified to be able to characterize in vivo fructose-1,6-diphosphate concentration. Subsequently, the biosensors were applied for fine-tuning glycolysis flux to effectively balance the biosynthesis of two chemicals: mevalonate and N-acetylglucosamine. A glycolysis flux-dynamically controlled Escherichia coli strain achieved a 111.3 g/L mevalonate titer in a 1L fermenter.     

Engineering the oleaginous yeast Yarrowia lipolytica for high-level resveratrol production

Resveratrol is a plant secondary metabolite with multiple health-beneficial properties. Microbial production of resveratrol in model microorganisms requires extensive engineering to reach commercially viable levels. Here, we explored the potential of the non-conventional yeast Yarrowia lipolytica to produce resveratrol and several other shikimate pathway-derived metabolites (p-coumaric acid, cis,cis-muconic acid, and salicylic acid). The Y. lipolytica strain expressing a heterologous pathway produced 52.1 ± 1.2 mg/L resveratrol in a small-scale cultivation. The titer increased to 409.0 ± 1.2 mg/L when the strain was further engineered with feedback-insensitive alleles of the key genes in the shikimate pathway and with five additional copies of the heterologous biosynthetic genes. In controlled fed-batch bioreactor, the strain produced 12.4 ± 0.3 g/L resveratrol, the highest reported titer to date for de novo resveratrol production, with a yield on glucose of 54.4 ± 1.6 mg/g and a productivity of 0.14 ± 0.01 g/L/h. The study showed that Y. lipolytica is an attractive host organism for the production of resveratrol and possibly other shikimate-pathway derived metabolites.     

Reconstitution of the peptidoglycan cytoplasmic precursor biosynthetic pathway in cell-free system and rapid screening of antisense oligonucleotides for Mur enzymes

Bacterial peptidoglycan is the cell wall component responsible for various biological activities. Its cytoplasmic precursor UDP-N-acetylmuramyl pentapeptide is biosynthesized by the first six enzymes of peptidoglycan synthetic pathways (Mur enzymes), which are all proved to be important targets for antibiotic screening. In our present work, the genes encoding Mur enzymes from Escherichia coli were co-expressed in the cell-free protein synthesis (CFPS) system, and the activities of Mur enzymes derived from CFPS system were validated by the synthesis of the final product UDP-N-acetylmuramyl pentapeptide. Then this in vitro reconstituted Mur biosynthetic pathway was used to screen a panel of specific antisense oligonucleotides for MurA and MurB. The selected oligonucleotides were proved to eliminate the expression of Mur enzymes, and thus inhibit the Mur biosynthetic pathway. The present work not only developed a rapid method to reconstruct and regulate a biosynthetic pathway in vitro, but also may provide insight into the development of novel antibiotics targeting on peptidoglycan biosynthetic pathway.     

A dopamine-based biosynthetic pathway produces decarboxylated betalains in Chenopodium quinoa

Betalains are the nitrogenous pigments that replace anthocyanins in the plant order Caryophyllales. Here, we describe unconventional decarboxylated betalains in quinoa (Chenopodium quinoa) grains. Decarboxylated betalains are derived from a previously unconsidered activity of the 4,5-DOPA-extradiol-dioxygenase enzyme (DODA), which has been identified as the key enzymatic step in the established biosynthetic pathway of betalains. Here, dopamine is fully characterized as an alternative substrate of the DODA enzyme able to yield an intermediate and structural unit of plant pigments: 6-decarboxy-betalamic acid, which is proposed and described. To characterize this activity, quinoa grains of different colors were analyzed in depth by chromatography, time-of-flight mass spectrometry, and reactions were performed in enzymatic assays and bioreactors. The enzymatic-chemical scheme proposed leads to an uncharacterized family of 6-decarboxylated betalains produced by a hitherto unknown enzymatic activity. All intermediate compounds as well as the final products of the dopamine-based biosynthetic pathway of pigments have been unambiguously determined and the reactions have been characterized from the enzymatic and functional perspectives. Results evidence a palette of molecules in quinoa grains of physiological relevance and which explain minor betalains described in plants of the Caryophyllales order. An entire family of betalains is anticipated.

A biosynthetic pathway produces unconventional plant pigments betalains derived from dopamine in quinoa.