Covalent Interactions of Anthocyanins with Proteins: Activity-Based Protein Profiling of Cyanidin-3-O-glucoside.

Anthocyanins are common natural pigments with a variety of physiological activities. Traditional perspectives attribute their molecular mechanism to noncovalent interactions influencing signaling pathways. However, this ignores the nature of its benzopyrylium skeleton, which readily reacts with the electron-rich groups of proteins. Here, we modified cyanidin-3-O-glucoside (C3G) via activity-based protein profiling technology by our previous synthesis route and prepared the covalent binding probe (C3G-Probe) and the noncovalent photoaffinity probe (C3G-Diazirine). The properties of C3G's covalent binding to proteins were also discovered by comparing the labeling of the two probes to the whole HepG2 cell proteome. We further explored its target proteins and enriched pathways in HepG2 and HeLa cells. Western blot analysis further confirmed the covalent binding of C3G to four target proteins: insulin-degrading enzyme, metal cation symporter ZIP14, spermatid perinuclear RNA-binding protein, and Cystatin-B. Pathway analysis showed that covalent targets of C3G were concentrated in metabolic pathways and several ribonucleoprotein complexes that were also coenriched. The results of this study provide new insights into the interaction of the naturally active molecule C3G with proteins.

Characterization of the covalent binding of cyanidin-3-glucoside to bovine serum albumin and its inhibition mechanism for advanced nonenzymatic glycosylation reactions.

Nonenzymatic glycosylation of proteins can generate advanced glycosylation end products, which are closely associated with the pathogenesis of certain chronic physiological diseases and aging. In this study, we characterized the covalent binding of cyanidin-3-glucoside (C3G) to bovine serum albumin (BSA) and investigated the mechanism by which this covalent binding inhibits the nonenzymatic glycosylation of BSA. The results indicated that the covalent interaction between C3G and BSA stabilized the protein's secondary structure. Through liquid chromatography-electrospray ionization tandem mass spectrometry analysis, we identified the covalent binding sites of C3G on BSA as lysine, arginine, asparagine, glutamine, and cysteine residues. This covalent interaction significantly suppressed the nonenzymatic glycosylation of BSA, consequently reducing the formation of nonenzymatic glycosylation products. C3G competitively binds to nonenzymatic glycosylation sites (e.g., lysine and arginine) on BSA, thereby impeding the glycosylation process and preventing the misfolding and structural alterations of BSA induced by fructose. Furthermore, the covalent attachment of C3G to BSA preserves the secondary structure of BSA and hinders subsequent nonenzymatic glycosylation events.

Effect of dapagliflozin on ferroptosis through the gut microbiota metabolite TMAO during myocardial ischemia-reperfusion injury in diabetes mellitus rats.

Dapagliflozin (DAPA) demonstrates promise in the management of diabetic mellitus (DM) and cardiomyopathy. Trimethylamine N-oxide (TMAO) is synthesized by the gut microbiota through the metabolic conversion of choline and phosphatidylcholine. Ferroptosis may offer novel therapeutic avenues for the management of diabetes and myocardial ischemia-reperfusion injury (IRI). However, the precise mechanism underlying ferroptosis in cardiomyocytes and the specific role of TMAO generated by gut microbiota in the therapeutic approach for DM and myocardial IRI utilizing DAPA need to be further explored. Nine male SD rats with specific pathogen-free (SPF) status were randomly divided equally into the normal group, the DM + IRI (DIR) group, and the DAPA group. The diversity of the gut microbiota was analyzed using 16S rRNA gene sequencing. Additionally, the Wekell technique was employed to measure the levels of TMAO in the three groups. Application of network pharmacology to search for intersection targets of DAPA, DIR, and ferroptosis, and RT-PCR experimental verification. Ultimately, the overlapping targets that were acquired were subjected to molecular docking analysis with TMAO. The changes of Bacteroidetes and Firmicutes in the gut microbiota of DIR rats were most significantly affected by DAPA. Escherichia-Shigella and Prevotella_9 within the phylum Bacteroidetes could be identified as the primary effects of DAPA on DIR. Compared with the normal group, the TMAO content in the DIR group was significantly increased, while the TMAO content in the DAPA group was decreased compared to the DIR group. For the network pharmacology analysis, DAPA and DIR generated 43 intersecting target genes, and then further intersected with ferroptosis-related genes, resulting in 11 overlapping target genes. The mRNA expression of ALB, HMOX1, PPARG, CBS, LCN2, and PPARA decreased in the DIR group through reverse transcription polymerase chain reaction (RT-PCR) validation, while the opposite trend was observed in the DAPA group. The docking score between TMAO and DPP4 was - 5.44, and the MM-GBSA result of - 22.02 kcal/mol. It epitomizes the finest docking performance among all the target genes with the lowest score. DAPA could reduce the levels of metabolite TMAO produced by gut microbiota, thereby regulating related target genes to decrease ferroptosis in DIR cardiomyocytes.

Elicitors directed in vitro growth and production of stevioside and other secondary metabolites in Stevia rebaudiana (Bertoni) Bertoni.

Stevia rebaudiana (stevia) is a plant in the Asteraceae that contains several biologically active compounds including the antidiabetic diterpene glycosides (e.g. stevioside, rebaudioside and dulcoside) that can serve as zero-calorie sugar alternatives. In this study, an elicitation strategy was applied using 5% polyethylene glycol (PEG), sodium chloride (NaCl; 50 and 100 mM) and gibberellic acid (2.0 and 4.0 mg/L GA3) to investigate their effect on shoot morphogenesis, and the production of phenolics, flavonoids, total soluble sugars, proline and stevioside, as well as antioxidant activity, in shoot cultures of S. rebaudiana. Herewith, the media supplemented with 2 mg/L and 4 mg/L GA3 exhibited the highest shooting response (87% and 80%). The augmentation of lower concentrations of GA3 (2 mg/L) in combination with 6-benzylaminopurine (BAP) resulted in the maximum mean shoot length (11.1 cm). The addition of 100 mM NaCl salts to the media led to the highest observed total phenolics content (TPC; 4.11 mg/g-DW compared to the control 0.52 mg/g-DW), total flavonoids content (TFC; 1.26 mg/g-DW) and polyphenolics concentration (5.39 mg/g-DW) in shoots cultured. However, the maximum antioxidant activity (81.8%) was observed in shoots raised in media treated with 50 mM NaCl. The application of 2 mg/L of GA3 resulted in the highest accumulation of proline (0.99 µg/mL) as compared to controls (0.37 µg/mL). Maximum stevioside content (71 µL/mL) was observed in cultures supplemented with 100 mM NaCl and 5% PEG, followed by the 4 mg/L GA3 treatment (70 µL/mL) as compared to control (60 µL/mL). Positive correlation was observed between GA3 and stevioside content. Notably, these two compounds are derived from a shared biochemical pathway. These results suggest that elicitation is an effective option to enhance the accumulation of steviosides and other metabolites and provides the groundwork for future industrial scale production using bioreactors.

Functional characterization of polyphenol oxidase OfPPO2 supports its involvement in parallel biosynthetic pathways of acteoside.

Acteoside is a bioactive phenylethanoid glycoside widely distributed throughout the plant kingdom. Because of its two catechol moieties, acteoside displays a variety of beneficial activities. The biosynthetic pathway of acteoside has been largely elucidated, but the assembly logic of two catechol moieties in acteoside remains unclear. Here, we identified a novel polyphenol oxidase OfPPO2 from Osmanthus fragrans, which could hydroxylate various monophenolic substrates, including tyrosine, tyrosol, tyramine, 4-hydroxyphenylacetaldehyde, salidroside, and osmanthuside A, leading to the formation of corresponding catechol-containing intermediates for acteoside biosynthesis. OfPPO2 could also convert osmanthuside B into acteoside, creating catechol moieties directly via post-modification of the acteoside skeleton. The reverse transcription-quantitative polymerase chain reaction (RT-qPCR) analysis and subcellular localization assay further support the involvement of OfPPO2 in acteoside biosynthesis in planta. These findings suggest that the biosynthesis of acteoside in O. fragrans may follow "parallel routes" rather than the conventionally considered linear route. In support of this hypothesis, the glycosyltransferase OfUGT and the acyltransferase OfAT could direct the flux of diphenolic intermediates generated by OfPPO2 into acteoside. Significantly, OfPPO2 and its orthologs constitute a functionally conserved enzyme family that evolved independently from other known biosynthetic enzymes of acteoside, implying that the substrate promiscuity of this PPO family may offer acteoside-producing plants alternative ways to synthesize acteoside. Overall, this work expands our understanding of parallel pathways plants may employ to efficiently synthesize acteoside, a strategy that may contribute to plants' adaptation to environmental challenges.

Efficient and novel biosynthesis of myricetin α-triglucoside with improved solubility using amylosucrase from Deinococcus deserti.

Although myricetin (3,3',4',5,5',7-hexahydroxyflavone, MYR) has a high antioxidant capacity and health functions, its use as a functional food material is limited owing to its low stability and water solubility. Amylosucrase (ASase) is capable of biosynthesizing flavonol α-glycoside using flavonols as acceptor molecules and sucrose as a donor molecule. Here, ASase from Deinococcus deserti (DdAS) efficiently biosynthesizes a novel MYR α-triglucoside (MYRαG3) using MYR as the acceptor molecule. Comparative homology analysis and computational simulation revealed that DdAS has a different active pocket for the transglycosylation reaction. DdAS produced MYRαG3 with a conversion efficiency of 67.4 % using 10 mM MYR and 50 mM sucrose as acceptor and donor molecules, respectively. The structure of MYRαG3 was identified as MYR 4'-O-4″,6″-tri-O-α-D-glucopyranoside using NMR and LC-MS. In silico analysis confirmed that DdAS has a distinct active pocket compared to other ASases. In addition, molecular docking simulations predicted the synthetic sequence of MYRαG3. Furthermore, MYRαG3 showed a similar DPPH radical scavenging activity of 49 %, comparable to MYR, but with significantly higher water solubility, which increased from 0.03 µg/mL to 511.5 mg/mL. In conclusion, this study demonstrated the efficient biosynthesis of a novel MYRαG3 using DdAS and highlighted the potential of MYRαG3 as a functional material.

Bioconversion of lignans in flaxseed cake by fermented tofu microbiota and isolation of Enterococcus faecium strain ZB26 responsible for converting secoisolariciresinol diglucoside to enterodiol.

Human intestinal microbiota plays a crucial role in converting secoisolariciresinol diglucoside, a lignan found in flaxseed, to enterodiol, which has a range of health benefits: antioxidative, antitumor, and estrogenic/anti-estrogenic effects. Given the high secoisolariciresinol diglucoside content in flaxseed cake, this study investigated the potential of co-fermenting flaxseed cake with fermented soybean product to isolate bacterial strains that effectively convert secoisolariciresinol diglucoside to enterodiol in a controlled environment (in vitro). The co-fermentation process with stinky tofu microbiota significantly altered the lignan, generating 12 intermediate lignan metabolites as identified by targeted metabolomics. One particular promising strain, ZB26, demonstrated an impressive ability to convert secoisolariciresinol diglucoside. It achieved a conversion rate of 87.42 ± 0.33%, with secoisolariciresinol and enterodiol generation rates of 94.22 ± 0.51% and 2.91 ± 0.03%, respectively. Further optimization revealed, under specific conditions (0.5 mM secoisolariciresinol diglucoside, pH 8, 30 °C for 3 days), ZB26 could convert an even higher percentage (97.75 ± 0.05%) of the secoisolariciresinol diglucoside to generate secoisolariciresinol (103.02 ± 0.16%) and enterodiol (3.18 ± 0.31%). These findings suggest that the identified strains ZB26 have promising potential for developing functional foods and ingredients enriched with lignans.

Assessment on malvidin-3-glucoside interaction with TLR4 via multi-spectroscopic analysis and molecular docking.

As one innate immune pattern recognition receptor, Toll-like receptor 4 (TLR4) recently has been considered as a critical player in glucolipid metabolism. Blueberries contain high level of anthocyanins, especially malvidin-3-glucoside (Mv-3-glc), which contribute the anti-inflammatory, hypoglycemic, and hypolipidemic effects. It is speculated that Mv-3-glc is able to possess these functions by binding to TLR4. Here, the noncovalent interactions of Mv-3-glc and TLR4 was explored through multi-techniques including fluorescence and ultraviolet-visible (UV-Vis) absorption spectroscopy, as well as molecular docking. The results demonstrated that Mv-3-glc was able to quench TLR4 intrinsic fluorescence effectively. A stable complex was formed spontaneously and the reaction was exothermic. The degree of binding of Mv-3-glc to TLR4 showed a strong dependence on the chemical concentration, temperature, and pH values. The negative signs for enthalpy (ΔH = -69.1 ± 10.8 kJ/mol) and entropy (ΔS = -105.0 ± 12.3 J/mol/K) from the interaction of the Mv-3-glc and TLR4 shows that the major driving forces are the hydrogen bonding and van der Waals' force, which is consistent with the molecular docking results. In addition, molecular docking predicted that the active center with specific amino acid residues, Phe126, Ser127, Leu54, Ile153, and Tyr131 was responsible for the site of Mv-3-glc binding to TLR4/myeloid differentiation protein-2 (MD-2). These findings confirmed that Mv-3-glc could bind to TLR4, which would be beneficial to understand the target therapeutic effects of blueberry anthocyanins on TLR4 in regulating glucolipid metabolism.

Accumulation patterns of anthocyanin and γ-oryzanol during black rice grain development.

Pigmented rice, especially black rice, is gaining popularity as it is rich in antioxidants such as anthocyanins and γ-oryzanol. At present, knowledge about temporal control of biosynthesis and accumulation of antioxidants during grain development is limited. To address this, the accumulation patterns of anthocyanins and γ-oryzanol were assessed in two distinct black rice genotypes over the course of grain development, and the expression of known regulatory genes for anthocyanin biosynthesis was examined. The results indicated that total γ-oryzanol content increased continuously throughout grain development, while total anthocyanins peaked at dough stage (15 to 21 days after flowering) followed by a decline until grain maturity in both genotypes. However, the rate of decrease in anthocyanin content differed between genotypes, and a more prominent decline in cyanidin 3-O-glucoside (C3G) relative to peonidin 3-O-glucoside (P3G) was observed for both. Anthocyanin content was closely linked with the expression of key regulatory genes in the MBW (MYB-bHLH-WD40) complex. This improved knowledge of the genotype-specific biosynthesis (anthocyanins only) and accumulation patterns of anthocyanins and γ-oryzanol can inform subsequent research efforts to increase concentrations of these key antioxidants in black rice grains.

A New Culture Medium Rich in Phenols Used for Screening Bitter Degrading Strains of Lactic Acid Bacteria to Employ in Table Olive Production.

The olive oil industry recently introduced a novel multi-phase decanter with the "Leopard DMF" series, which gives a by-product called pâté, made up of pulp and olive wastewater with a high content of phenolic substances and without pits. This study aims to create a new culture medium, the Olive Juice Broth (OJB), from DMF pâté, and apply it to select bacteria strains able to survive and degrade the bitter substances normally present in the olive fruit. Thirty-five different bacterial strains of Lactiplantibacillus plantarum from the CREA-IT.PE Collection of Microorganisms were tested. Seven strains characterized by ≥50% growth in OJB (B31, B137, B28, B39, B124, B130, and B51) showed a degradation of the total phenolic content of OJB ≥ 30%. From this set, L. plantarum B51 strain was selected as a starter for table olive production vs. spontaneous fermentation. The selected inoculant effectively reduced the debittering time compared to spontaneous fermentation. Hydroxytyrosol, derived from oleuropein and verbascoside degradation, and tyrosol, derived from ligstroside degradation, were produced faster than during spontaneous fermentation. The OJB medium is confirmed to be useful in selecting bacterial strains resistant to the complex phenolic environment of the olive fruit.

Independent regulation of strigolactones and blumenols during arbuscular mycorrhizal symbiosis in rice.

The apocarotenoid strigolactones (SLs) facilitate pre-symbiotic communication between arbuscular mycorrhizal (AM) fungi and plants. Related blumenol-C-glucosides (blumenols), have also been associated with symbiosis, but the cues that are involved in the regulation of blumenol accumulation during AM symbiosis remain unclear. In rice, our analyses demonstrated a strict correlation between foliar blumenol abundance and intraradical fungal colonisation. More specifically, rice mutants affected at distinct stages of the interaction revealed that fungal cortex invasion was required for foliar blumenol accumulation. Plant phosphate status and D14L hormone signalling had no effect, contrasting their known role in induction of SLs. This a proportion of the SL biosynthetic enzymes, D27 and D17, are equally required for blumenol production. These results importantly clarify that, while there is a partially shared biosynthetic pathway between SL and blumenols, the dedicated induction of the related apocarotenoids occurs in response to cues acting at distinct stages during the root colonisation process. However, we reveal that neither SLs nor blumenols are essential for fungal invasion of rice roots.

Development of a gene-coded biosensor to establish a high-throughput screening platform for salidroside production.

Metabolic engineering reconfigures cellular networks to produce value-added compounds from renewable substrates efficiently. However, identifying strains with desired phenotypes from large libraries through rational or random mutagenesis remains challenging. To overcome this bottleneck, an effective high-throughput screening (HTS) method must be developed to detect and analyze target candidates rapidly. Salidroside is an aromatic compound with broad applications in food, healthcare, medicine, and daily chemicals. However, there currently needs to be HTS methods available to monitor salidroside levels or to screen enzyme variants and strains for high-yield salidroside biosynthesis, which severely limits the development of microbial cell factories capable of efficiently producing salidroside on an industrial scale. This study developed a gene-encoded whole-cell biosensor that is specifically responsive to salidroside. The biosensor was created by screening a site-saturated mutagenic library of uric acid response regulatory protein binding bags. This work demonstrates the feasibility of monitoring metabolic flux with whole-cell biosensors for critical metabolites. It provides a promising tool for building salidroside high-yielding strains for high-throughput screening and metabolic regulation to meet industrial needs.

Biosynthesis of Apigenin Glucosides in Engineered Corynebacterium glutamicum.

Glucosylation is a well-known approach to improve the solubility, pharmacological, and biological properties of flavonoids, making flavonoid glucosides a target for large-scale biosynthesis. However, the low yield of products coupled with the requirement of expensive UDP-sugars limits the application of enzymatic systems for large-scale. C. glutamicum is a Gram-positive and generally regarded as safe (GRAS) bacteria frequently employed for the large-scale production of amino acids and bio-fuels. Due to the versatility of its cell factory system and its non-endotoxin producing properties, it has become an attractive system for the industrial-scale biosynthesis of alternate products. Here, we explored the cell factory of C. glutamicum for efficient glucosylation of flavonoids using apigenin as a model flavonoid, with the heterologous expression of a promiscuous glycosyltransferase, YdhE from Bacillus licheniformis and the endogenous overexpression of C. glutamicum genes galU1 encoding UDP-glucose pyrophosphorylase and pgm encoding phosphoglucomutase involved in the synthesis of UDP-glucose to create a C. glutamicum cell factory system capable of efficiently glucosylation apigenin with a high yield of glucosides production. Consequently, the production of various apigenin glucosides was controlled under different temperatures yielding almost 4.2 mM of APG1(apigenin-4'-O-ß-glucoside) at 25°C, and 0.6 mM of APG2 (apigenin-7-O-ß-glucoside), 1.7 mM of APG3 (apigenin-4',7-O-ß-diglucoside) and 2.1 mM of APG4 (apigenin-4',5-O-ß-diglucoside) after 40 h of incubation with the supplementation of 5 mM of apigenin and 37°C. The cost-effective developed system could be used to modify a wide range of plant secondary metabolites with increased pharmacokinetic activities on a large scale without the use of expensive UDP-sugars.

Disentangling hydroxynitrile glucoside biosynthesis in a barley (Hordeum vulgare) metabolon provides access to elite malting barleys for ethyl carbamate-free whisky production.

Barley produces several specialized metabolites, including five α-, ß-, and γ-hydroxynitrile glucosides (HNGs). In malting barley, presence of the α-HNG epiheterodendrin gives rise to undesired formation of ethyl carbamate in the beverage production, especially after distilling. Metabolite-GWAS identified QTLs and underlying gene candidates possibly involved in the control of the relative and absolute content of HNGs, including an undescribed MATE transporter. By screening 325 genetically diverse barley accessions, we discovered three H. vulgare ssp. spontaneum (wild barley) lines with drastic changes in the relative ratios of the five HNGs. Knock-out (KO)-lines, isolated from the barley FIND-IT resource and each lacking one of the functional HNG biosynthetic genes (CYP79A12, CYP71C103, CYP71C113, CYP71U5, UGT85F22 and UGT85F23) showed unprecedented changes in HNG ratios enabling assignment of specific and mutually dependent catalytic functions to the biosynthetic enzymes involved. The highly similar relative ratios between the five HNGs found across wild and domesticated barley accessions indicate assembly of the HNG biosynthetic enzymes in a metabolon, the functional output of which was reconfigured in the absence of a single protein component. The absence or altered ratios of the five HNGs in the KO-lines did not change susceptibility to the fungal phytopathogen Pyrenophora teres causing net blotch. The study provides a deeper understanding of the organization of HNG biosynthesis in barley and identifies a novel, single gene HNG-0 line in an elite spring barley background for direct use in breeding of malting barley, eliminating HNGs as a source of ethyl carbamate formation in whisky production.

Efficient biotransformation of naringenin to naringenin α-glucoside, a novel α-glucosidase inhibitor, by amylosucrase from Deinococcus wulumuquiensis.

Amylosucrase (ASase) efficiently biosynthesizes α-glucoside using flavonoids as acceptor molecules and sucrose as a donor molecule. Here, ASase from Deinococcus wulumuqiensis (DwAS) biosynthesized more naringenin α-glucoside (NαG) with sucrose and naringenin as donor and acceptor molecules, respectively, than other ASases from Deinococcus sp. The biotransformation rate of DwAS to NαG was 21.3% compared to 7.1-16.2% for other ASases. Docking simulations showed that the active site of DwAS was more accessible to naringenin than those of others. The 217th valine in DwAS corresponded to the 221st isoleucine in Deinococcus geothermalis AS (DgAS), and the isoleucine possibly prevented naringenin from accessing the active site. The DwAS-V217I mutant had a significantly lower biosynthetic rate of NαG than DwAS. The kcat/Km value of DwAS with naringenin as the donor was significantly higher than that of DgAS and DwAS-V217I. In addition, NαG inhibited human intestinal α-glucosidase more efficiently than naringenin.

Enhancing Gastrodin Production in Yarrowia lipolytica by Metabolic Engineering.

Gastrodin, 4-hydroxybenzyl alcohol-4-O-ß-D-glucopyranoside, has been widely used in the treatment of neurogenic and cardiovascular diseases. Currently, gastrodin biosynthesis is being achieved in model microorganisms. However, the production levels are insufficient for industrial applications. In this study, we successfully engineered a Yarrowia lipolytica strain to overproduce gastrodin through metabolic engineering. Initially, the engineered strain expressing the heterologous gastrodin biosynthetic pathway, which comprises chorismate lyase, carboxylic acid reductase, phosphopantetheinyl transferase, endogenous alcohol dehydrogenases, and a UDP-glucosyltransferase, produced 1.05 g/L gastrodin from glucose in a shaking flask. Then, the production was further enhanced to 6.68 g/L with a productivity of 2.23 g/L/day by overexpressing the key node DAHP synthases of the shikimate pathway and alleviating the native tryptophan and phenylalanine biosynthetic pathways. Finally, the best strain, Gd07, produced 13.22 g/L gastrodin in a 5 L fermenter. This represents the highest reported production of gastrodin in an engineered microorganism to date, marking the first successful de novo production of gastrodin using Y. lipolytica.

Antagonistic properties against Fusarium sporotrichioides and glycosylation of HT-2 and T-2 toxins by selected Trichoderma strains.

The present study assessed the ability of Trichoderma to combat F. sporotrichioides, focusing on their antagonistic properties. Tests showed that Trichoderma effectively inhibited F. sporotrichioides mycelial growth, particularly with T. atroviride strains. In co-cultures on rice grains, Trichoderma almost completely reduced the biosynthesis of T-2 and HT-2 toxins by Fusarium. T-2 toxin-α-glucoside (T-2-3α-G), HT-2 toxin-α-glucoside (HT-2-3α-G), and HT-2 toxin-ß-glucoside (HT-2-3ß-G) were observed in the common culture medium, while these substances were not present in the control medium. The study also revealed unique metabolites and varying metabolomic profiles in joint cultures of Trichoderma and Fusarium, suggesting complex interactions. This research offers insights into the processes of biocontrol by Trichoderma, highlighting its potential as a sustainable solution for managing cereal plant pathogens and ensuring food safety.

Leveraging a Y. lipolytica naringenin chassis for biosynthesis of apigenin and associated glucoside.

Flavonoids are a diverse set of natural products with promising bioactivities including anti-inflammatory, anti-cancer, and neuroprotective properties. Previously, the oleaginous host Yarrowia lipolytica has been engineered to produce high titers of the base flavonoid naringenin. Here, we leverage this host along with a set of E. coli bioconversion strains to produce the flavone apigenin and its glycosylated derivative isovitexin, two potential nutraceutical and pharmaceutical candidates. Through downstream strain selection, co-culture optimization, media composition, and mutant isolation, we were able to produce168 mg/L of apigenin, representing a 46% conversion rate of 2-(R/S)-naringenin to apigenin. This apigenin platform was modularly extended to produce isovitexin by addition of a second bioconversion strain. Together, these results demonstrate the promise of microbial production and modular bioconversion to access diversified flavonoids.

One-pot sustainable synthesis of glucosylglycerate from starch and glycerol through artificial in vitro enzymatic cascade.

Glucosylglycerate (R-2-O-α-D-glucopyranosyl-glycerate, GG) is a negatively charged compatible solution with versatile functions. Here, an artificial in vitro enzymatic cascade was designed to feasibly and sustainably produce GG from affordable starch and glycerol. First, Spirochaeta thermophila glucosylglycerate phosphorylase (GGP) was carefully selected because of its excellent heterologous expression, specific activity, and thermostability. The optimized two-enzyme cascade, consisting of alpha-glucan phosphorylase (αGP) and GGP, achieved a remarkable 81 % conversion rate from maltodextrin and D-glycerate. Scaling up this cascade resulted in a practical concentration of 58 g/L GG with a 62 % conversion rate based on the added D-glycerate. Additionally, the production of GG from inexpensive starch and glycerol in one-pot using artificial four-enzyme cascade was successfully implemented, which integrates alditol oxidase and catalase with αGP and GGP. Collectively, this sustainable enzymatic cascade demonstrates the feasibility of the practical synthesis of GG and has the potential to produce other glycosides using the phosphorylase-and-phosphorylase paradigm.

Cloning, Expression, and Functional Characterization of Two Highly Efficient Flavonoid-di-O-glycosyltransferases ZmUGT84A1 and ZmUGT84A2 from Maize (Zea mays L.).

The maize (Zea mays L.) glycosyltransferase family 1 comprises many uridine diphosphate glycosyltransferase (UGT) members. However, UGT activities and biochemical functions have seldom been revealed. In this study, the genes of two flavonoid di-O-glycosyltransferases ZmUGT84A1 and ZmUGT84A2 were cloned from maize plant and expressed in Escherichia coli. Phylogenetic analysis showed that the two enzymes were homologous to AtUGT84A1 and AtUGT84A3. The two recombinant enzymes showed a high conversion rate of luteolin to its glucosides, mainly 4',7-di-O-glucoside and minorly 3',7-di-O-glucoside in two-step glycosylation reactions in vitro. Moreover, the recombinant ZmUGT84A1 and ZmUGT84A2 had a broad substrate spectrum, converting eriodictyol, naringenin, apigenin, quercetin, and kaempferol to monoglucosides and diglucosides. The highly efficient ZmUGT84A1 and ZmUGT84A2 may be used as a tool for the effective synthesis of various flavonoid O-glycosides and as markers for crop breeding to increase O-glycosyl flavonoid content in food.