De Novo Production of Glycyrrhetic Acid 3-O-mono-β-D-glucuronide in Saccharomyces cerevisiae.
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ABSTRACT:
SUBMITTER: Huang Y
PROVIDER: S-EPMC8650490 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Glycyrrhizin (GL) and Glycyrrhetic Acid 3-O-mono-β-D-glucuronide (GAMG) are the typical triterpenoid glycosides found in the root of licorice, a popular medicinal plant that exhibits diverse physiological effects and pharmacological manifestations. However, only few reports are available on the glycosylation enzymes involved in the biosynthesis of these valuable compounds with low conversion yield so far. In mammals, glycosyltransferases are involved in the phase II metabolism and may provide new solutions for us to engineer microbial strains to produce high valued compounds due to the substrate promiscuity of these glycosyltransferases. In this study, we mined the genomic databases of mammals and evaluated 22 candidate genes of O-glycosyltransferases by analyzing their catalytic potential for O-glycosylation of the native substrate, glycyrrhetinic acid (GA) for its glycodiversification. Out of 22 selected glycosyltransferases, only UGT1A1 exhibited high catalytic performance for biosynthesis of the key licorice compounds GL and GAMG. Molecular docking results proposed that the enzymatic activity of UGT1A1 was likely owing to the stable hydrogen bonding interactions and favorite conformations between the amino acid residues around substrate channels (P82~R85) and substrates. Furthermore, the complete biosynthesis pathway of GL was reconstructed in Saccharomyces cerevisiae for the first time, resulting in the production of 5.98 ± 0.47 mg/L and 2.31 ± 0.21 mg/L of GL and GAMG, respectively.
| S-EPMC8283272 | biostudies-literature
Project description:BackgroundAnthocyanins are polyphenolic pigments which provide pink to blue colours in fruits and flowers. There is an increasing demand for anthocyanins, as food colorants and as health-promoting substances. Plant production of anthocyanins is often seasonal and cannot always meet demand due to low productivity and the complexity of the plant extracts. Therefore, a system of on-demand supply is useful. While a number of other (simpler) plant polyphenols have been successfully produced in the yeast Saccharomyces cerevisiae, production of anthocyanins has not yet been reported.ResultsSaccharomyces cerevisiae was engineered to produce pelargonidin 3-O-glucoside starting from glucose. Specific anthocyanin biosynthetic genes from Arabidopsis thaliana and Gerbera hybrida were introduced in a S. cerevisiae strain producing naringenin, the flavonoid precursor of anthocyanins. Upon culturing, pelargonidin and its 3-O-glucoside were detected inside the yeast cells, albeit at low concentrations. A number of related intermediates and side-products were much more abundant and were secreted into the culture medium. To optimize titers of pelargonidin 3-O-glucoside further, biosynthetic genes were stably integrated into the yeast genome, and formation of a major side-product, phloretic acid, was prevented by engineering the yeast chassis. Further engineering, by removing two glucosidases which are known to degrade pelargonidin 3-O-glucoside, did not result in higher yields of glycosylated pelargonidin. In aerated, pH controlled batch reactors, intracellular pelargonidin accumulation reached 0.01 µmol/gCDW, while kaempferol and dihydrokaempferol were effectively exported to reach extracellular concentration of 20 µM [5 mg/L] and 150 µM [44 mg/L], respectively.ConclusionThe results reported in this study demonstrate the proof-of-concept that S. cerevisiae is capable of de novo production of the anthocyanin pelargonidin 3-O-glucoside. Furthermore, while current conversion efficiencies are low, a number of clear bottlenecks have already been identified which, when overcome, have huge potential to enhance anthocyanin production efficiency. These results bode very well for the development of fermentation-based production systems for specific and individual anthocyanin molecules. Such systems have both great scientific value for identifying and characterising anthocyanin decorating enzymes as well as significant commercial potential for the production of, on-demand, pure bioactive compounds to be used in the food, health and even pharma industries.
| S-EPMC6029064 | biostudies-literature
Project description:To understand that 18β-Glycyrrhetic acid 3-O-mono-β-D-glucuronide (GAMG) showed better pharmacological activity and drug-like properties than 18β-Glycyrrhizin (GL); a rapid and sensitive HPLC-MS/MS method was established for the simultaneous determination of GAMG and its metabolite 18β-Glycyrrhetinic acid (GA) in rat plasma and tissues after oral administration of GAMG or GL. This analytical method was validated by linearity, LLOQ, specificity, recovery rate, matrix effect, etc. After oral administration, GAMG exhibited excellent Cmax (2377.57 ng/mL), Tmax (5 min) and AUC0-T (6625.54 mg/L*h), which was much higher than the Cmax (346.03 ng/mL), Tmax (2.00 h) and AUC0-T (459.32 mg/L*h) of GL. Moreover, GAMG had wider and higher tissue distribution in the kidney, spleen, live, lung, brain, etc. These results indicated that oral GAMG can be rapidly and efficiently absorbed and be widely distributed in tissues to exert stronger and multiple pharmacological activities. This provided a physiological basis for guiding the pharmacodynamic study and clinical applications of GAMG.
| S-EPMC9315563 | biostudies-literature
Project description:BACKGROUND: Flavonoids comprise a large family of secondary plant metabolic intermediates that exhibit a wide variety of antioxidant and human health-related properties. Plant production of flavonoids is limited by the low productivity and the complexity of the recovered flavonoids. Thus to overcome these limitations, metabolic engineering of specific pathway in microbial systems have been envisaged to produce high quantity of a single molecules. RESULT: Saccharomyces cerevisiae was engineered to produce the key intermediate flavonoid, naringenin, solely from glucose. For this, specific naringenin biosynthesis genes from Arabidopsis thaliana were selected by comparative expression profiling and introduced in S. cerevisiae. The sole expression of these A. thaliana genes yielded low extracellular naringenin concentrations (<5.5 ?M). To optimize naringenin titers, a yeast chassis strain was developed. Synthesis of aromatic amino acids was deregulated by alleviating feedback inhibition of 3-deoxy-d-arabinose-heptulosonate-7-phosphate synthase (Aro3, Aro4) and byproduct formation was reduced by eliminating phenylpyruvate decarboxylase (Aro10, Pdc5, Pdc6). Together with an increased copy number of the chalcone synthase gene and expression of a heterologous tyrosine ammonia lyase, these modifications resulted in a 40-fold increase of extracellular naringenin titers (to approximately 200 ?M) in glucose-grown shake-flask cultures. In aerated, pH controlled batch reactors, extracellular naringenin concentrations of over 400 ?M were reached. CONCLUSION: The results reported in this study demonstrate that S. cerevisiae is capable of de novo production of naringenin by coexpressing the naringenin production genes from A. thaliana and optimization of the flux towards the naringenin pathway. The engineered yeast naringenin production host provides a metabolic chassis for production of a wide range of flavonoids and exploration of their biological functions.
| S-EPMC3539886 | biostudies-literature
Project description:Glycyrrhetic acid 3-O-mono-β-d-glucuronide (GAMG) as an important derivative of glycyrrhizin (GL) shows stronger biological activities and higher sweetness than GL. The biotransformation process is considered as an efficient strategy for GAMG production, due to its mild reaction, high production efficiency and environmentally friendly status. In this study, licorice straw was used for the first time as a medium for GAMG and lignocellulosic enzyme production via solid-state fermentation (SSF) of endophytic fungus Chaetomium globosum DX-THS3. The fermentation conditions including particle size, temperature, seed age, inoculum size, and moisture of substrate were optimized. Furthermore, additional nitrogen sources and carbon sources were screened for GAMG production by C. globosum DX-THS3 of SSF. Under optimal fermentation conditions, the percent conversion of glycyrrhizin reached 90% in 15 days, whereas the control needed 35 days to achieve the same result. The productivity of optimization (P = 2.1 mg/g/day) was 2.33-fold that of non-optimization (P = 0.9 mg/g/day). Meanwhile, high activities of filter paper enzyme (FPase) (245.80 U/g), carboxymethyl cellulase (CMCase) (33.67 U/g), xylanase (83.44 U/g), and β-glucuronidase activity (271.42 U/g) were obtained faster than those in the control during SSF. Our study provides a novel and efficient strategy for GAMG production and indicates C. globosum DX-THS3 as a potential producer of lignocellulolytic enzymes.Supplementary informationThe online version contains supplementary material available at 10.1186/s40643-021-00441-y.
| S-EPMC8442819 | biostudies-literature
Project description:BACKGROUND:(+)-Nootkatone is a highly valued sesquiterpenoid compound, exhibiting a typical grapefruit aroma and various desired biological activities for use as aromatics and pharmaceuticals. The high commercial demand of (+)-nootkatone is predominately met by chemical synthesis, which entails the use of environmentally harmful reagents. Efficient synthesis of (+)-nootkatone via biotechnological approaches is thus urgently needed to satisfy its industrial demand. However, there are only a limited number of studies that report the de novo synthesis of (+)-nootkatone from simple carbon sources in microbial cell factories, and with relatively low yield. RESULTS:As the direct precursor of (+)-nootkatone biosynthesis, (+)-valencene was first produced in large quantities in Saccharomyces cerevisiae by overexpressing (+)-valencene synthase CnVS of Callitropsis nootkatensis in combination with various mevalonate pathway (MVA) engineering strategies, including the expression of CnVS and farnesyl diphosphate synthase (ERG20) as a fused protein, overexpression of a truncated form of the rate-limiting enzyme 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase (tHMG1), and downregulating the squalene synthase enzyme (ERG9). These approaches altogether brought the production of (+)-valencene to 217.95 mg/L. Secondly, we addressed the (+)-valencene oxidation by overexpressing the Hyoscyamus muticus premnaspirodiene oxygenase (HPO) variant (V482I/A484I) and cytochrome P450 reductase (ATR1) from Arabidopsis thaliana. However, (+)-valencene was predominantly oxidized to β-nootkatol and only minor amounts of (+)-nootkatone (9.66 mg/L) were produced. We further tackled the oxidation of β-nootkatol to (+)-nootkatone by screening various dehydrogenases. Our results showed that the short-chain dehydrogenase/reductase (SDR) superfamily dehydrogenases ZSD1 of Zingiber zerumbet and ABA2 of Citrus sinensis were capable of effectively catalyzing β-nootkatol oxidation to (+)-nootkatone. The yield of (+)-nootkatone increased to 59.78 mg/L and 53.48 mg/L by additional overexpression of ZSD1 and ABA2, respectively. CONCLUSION:We successfully constructed the (+)-nootaktone biosynthesis pathway in S. cerevisiae by overexpressing the (+)-valencene synthase CnVS, cytochrome P450 monooxygenase HPO, and SDR family dehydrogenases combined with the MVA pathway engineering, providing a solid basis for the whole-cell production of (+)-nootkatone. The two effective SDR family dehydrogenases tested in this study will serve as valuable enzymatic tools in further optimizing (+)-nootkatone production.
| S-EPMC6998195 | biostudies-literature
Project description:Monogalactosyldiacylglycerol (MGDG), a predominant photosynthetic membrane lipid derived from plants and microalgae, has important applications in feed additives, medicine, and other fields. The low content and various structural stereoselectivity differences of MGDG in plants limited the biological extraction or chemical synthesis of MGDG, resulting in a supply shortage of monogalactosyldiacylglycerol with a growing demand. Herein, we established Saccharomyces cerevisiae as a cell factory for efficient de novo production of monogalactosyldiacylglycerol for the first time. Heterologous production of monogalactosyldiacylglycerol was achieved by overexpression of codon-optimized monogalactosyldiacylglycerol synthase gene MGD1, the key Kennedy pathway genes (i.e. GAT1, ICT1, and PAH1), and multi-copy integration of the MGD1 expression cassette. The final engineered strain (MG-8) was capable of producing monogalactosyldiacylglycerol with titers as high as 16.58 nmol/mg DCW in a shake flask and 103.2 nmol/mg DCW in a 5 L fed-batch fermenter, respectively. This is the first report of heterologous biosynthesis of monogalactosyldiacylglycerol in microorganisms, which will provide a favorable reference for study on heterologous production of monogalactosyldiacylglycerol in yeasts.
| S-EPMC11318150 | biostudies-literature
Project description:BackgroundResveratrol is a plant secondary metabolite with diverse, potential health-promoting benefits. Due to its nutraceutical merit, bioproduction of resveratrol via microbial engineering has gained increasing attention and provides an alternative to unsustainable chemical synthesis and straight extraction from plants. However, many studies on microbial resveratrol production were implemented with the addition of water-insoluble phenylalanine or tyrosine-based precursors to the medium, limiting in the sustainable development of bioproduction.ResultsHere we present a novel coculture platform where two distinct metabolic background species were modularly engineered for the combined total and de novo biosynthesis of resveratrol. In this scenario, the upstream Escherichia coli module is capable of excreting p-coumaric acid into the surrounding culture media through constitutive overexpression of codon-optimized tyrosine ammonia lyase from Trichosporon cutaneum (TAL), feedback-inhibition-resistant 3-deoxy-d-arabinoheptulosonate-7-phosphate synthase (aroGfbr) and chorismate mutase/prephenate dehydrogenase (tyrAfbr) in a transcriptional regulator tyrR knockout strain. Next, to enhance the precursor malonyl-CoA supply, an inactivation-resistant version of acetyl-CoA carboxylase (ACC1S659A,S1157A) was introduced into the downstream Saccharomyces cerevisiae module constitutively expressing codon-optimized 4-coumarate-CoA ligase from Arabidopsis thaliana (4CL) and resveratrol synthase from Vitis vinifera (STS), and thus further improve the conversion of p-coumaric acid-to-resveratrol. Upon optimization of the initial inoculation ratio of two populations, fermentation temperature, and culture time, this co-culture system yielded 28.5 mg/L resveratrol from glucose in flasks. In further optimization by increasing initial net cells density at a test tube scale, a final resveratrol titer of 36 mg/L was achieved.ConclusionsThis is first study that demonstrates the use of a synthetic E. coli-S. cerevisiae consortium for de novo resveratrol biosynthesis, which highlights its potential for production of other p-coumaric-acid or resveratrol derived biochemicals.
| S-EPMC7362445 | biostudies-literature
Project description:BackgroundThe ideal biofuel should not only be a regenerative fuel from renewable feedstocks, but should also be compatible with the existing fuel distribution infrastructure and with normal car engines. As the so-called drop-in biofuel, the fatty alcohol 1-octanol has been described as a valuable substitute for diesel and jet fuels and has already been produced fermentatively from sugars in small amounts with engineered bacteria via reduction of thioesterase-mediated premature release of octanoic acid from fatty acid synthase or via a reversal of the β-oxidation pathway.ResultsThe previously engineered short-chain acyl-CoA producing yeast Fas1R1834K/Fas2 fatty acid synthase variant was expressed together with carboxylic acid reductase from Mycobacterium marinum and phosphopantetheinyl transferase Sfp from Bacillus subtilis in a Saccharomyces cerevisiae Δfas1 Δfas2 Δfaa2 mutant strain. With the involvement of endogenous thioesterases, alcohol dehydrogenases, and aldehyde reductases, the synthesized octanoyl-CoA was converted to 1-octanol up to a titer of 26.0 mg L-1 in a 72-h fermentation. The additional accumulation of 90 mg L-1 octanoic acid in the medium indicated a bottleneck in 1-octanol production. When octanoic acid was supplied externally to the yeast cells, it could be efficiently converted to 1-octanol indicating that re-uptake of octanoic acid across the plasma membrane is not limiting. Additional overexpression of aldehyde reductase Ahr from Escherichia coli nearly completely prevented accumulation of octanoic acid and increased 1-octanol titers up to 49.5 mg L-1. However, in growth tests concentrations even lower than 50.0 mg L-1 turned out to be inhibitory to yeast growth. In situ extraction in a two-phase fermentation with dodecane as second phase did not improve growth, indicating that 1-octanol acts inhibitive before secretion. Furthermore, 1-octanol production was even reduced, which results from extraction of the intermediate octanoic acid to the organic phase, preventing its re-uptake.ConclusionsBy providing chain length control via an engineered octanoyl-CoA producing fatty acid synthase, we were able to specifically produce 1-octanol with S. cerevisiae. Before metabolic engineering can be used to further increase product titers and yields, strategies must be developed that cope with the toxic effects of 1-octanol on the yeast cells.
| S-EPMC5984327 | biostudies-literature
Project description:Psilocybin is a tryptamine-derived psychoactive alkaloid found mainly in the fungal genus Psilocybe, among others, and is the active ingredient in so-called "magic mushrooms". Although its notoriety originates from its psychotropic properties and popular use as a recreational drug, clinical trials have recently recognized psilocybin as a promising candidate for the treatment of various psychological and neurological afflictions. In this work, we demonstrate the de novo biosynthetic production of psilocybin and related tryptamine derivatives in Saccharomyces cerevisiae by expression of a heterologous biosynthesis pathway sourced from Psilocybe cubensis. Additionally, we achieve improved product titers by supplementing the pathway with a novel cytochrome P450 reductase from P. cubensis. Further rational engineering resulted in a final production strain producing 627 ± 140 mg/L of psilocybin and 580 ± 276 mg/L of the dephosphorylated degradation product psilocin in triplicate controlled fed-batch fermentations in minimal synthetic media. Pathway intermediates baeocystin, nor norbaeocystin as well the dephosphorylated baeocystin degradation product norpsilocin were also detected in strains engineered for psilocybin production. We also demonstrate the biosynthetic production of natural tryptamine derivative aeruginascin as well as the production of a new-to-nature tryptamine derivative N-acetyl-4-hydroxytryptamine. These results lay the foundation for the biotechnological production of psilocybin in a controlled environment for pharmaceutical applications, and provide a starting point for the biosynthetic production of other tryptamine derivatives of therapeutic relevance.
| S-EPMC7232020 | biostudies-literature