Biotransformation approach to produce rare ginsenosides F1, compound Mc1, and Rd2 from major ginsenosides.

The stems and leaves of Panax notoginseng contain high saponins, but they are often discarded as agricultural waste. In this study, the predominant ginsenosides Rg1, Rc, and Rb2, presented in the stems and leaves of ginseng plants, were biotransformed into value-added rare ginsenosides F1, compound Mc1 (C-Mc1), and Rd2, respectively. A fungal strain YMS6 (Penicillium sp.) was screened from the soil as a biocatalyst with high selectivity for the deglycosylation of major ginsenosides. Under the optimal fermentation conditions, the yields of F1, C-Mc1, and Rd2 were 97.95, 68.64, and 79.58%, respectively. This study provides a new microbial resource for the selective conversion of protopanaxadiol-type and protopanaxatriol-type major saponins into rare ginsenosides via the whole-cell biotransformation and offers a solution for the better utilization of P. notoginseng waste.

Efficient biosynthesis of resveratrol via combining phenylalanine and tyrosine pathways in Saccharomyces cerevisiae.

BACKGROUND: Resveratrol is a commercially available stilbenoid widely used as dietary supplements, functional food ingredients, and cosmetic ingredients due to its diverse physiological activities. The production of resveratrol in microorganisms provides an ideal source that reduces the cost of resveratrol, but the titer in Saccharomyces cerevisiae was still much lower than that in other hosts. RESULTS: To achieve enhanced production of resveratrol in S. cerevisiae, we constructed a biosynthetic pathway via combining phenylalanine and tyrosine pathways by introducing a bi-functional phenylalanine/tyrosine ammonia lyase from Rhodotorula toruloides. The combination of phenylalanine pathway with tyrosine pathway led to a 462% improvement of resveratrol production in yeast extract peptone dextrose (YPD) medium with 4% glucose, suggesting an alternative strategy for producing p-coumaric acid-derived compounds. Then the strains were further modified by integrating multi-copy biosynthetic pathway genes, improving metabolic flux to aromatic amino acids and malonyl-CoA, and deleting by-pathway genes, which resulted in 1155.0 mg/L resveratrol in shake flasks when cultured in YPD medium. Finally, a non-auxotrophic strain was tailored for resveratrol production in minimal medium without exogenous amino acid addition, and the highest resveratrol titer (4.1 g/L) ever reported was achieved in S. cerevisiae to our knowledge. CONCLUSIONS: This study demonstrates the advantage of employing a bi-functional phenylalanine/tyrosine ammonia lyase in the biosynthetic pathway of resveratrol, suggesting an effective alternative in the production of p-coumaric acid-derived compounds. Moreover, the enhanced production of resveratrol in S. cerevisiae lays a foundation for constructing cell factories for various stilbenoids.

Biotransformation of Sclareol by a Fungal Endophyte of Salvia sclarea.

Microbial endophytes are known as versatile producers of useful metabolites, which have extensive applications in pharmacy, fragrance, agriculture and food. This study aims to screen sclareol-biotransforming microorganisms from Salvia sclarea, an untapped source of diverse endophytes. In this study, 50 culturable endophytes were isolated from S. sclarea grown in Xinjiang using sclareol as the sole carbon source and screened for their potential to transform sclareol into analogues. A fungal endophyte, identified as the generally recognized as safe (GRAS) strain Aspergillus tubingensis, can produce labd-14-ene-3ß,8α,13ß-triol and 8α,13ß-dihydroxylabd-14-en-3-one from sclareol, involving hydroxylation and carbonylation at the C3 site. Structures of the two metabolites were elucidated by HR-ESI-MS and NMR analysis. S. sclarea was proven to be a good source of endophytes that are prospective producers of secondary metabolites with valuable chemical and biological properties. This study is the first report regarding the isolation of endophytes from S. sclarea.

High-yield production of protopanaxadiol from sugarcane molasses by metabolically engineered Saccharomyces cerevisiae.

BACKGROUND: Ginsenosides are Panax plant-derived triterpenoid with wide applications in cardiovascular protection and immunity-boosting. However, the saponins content of Panax plants is fairly low, making it time-consuming and unsustainable by direct extraction. Protopanaxadiol (PPD) is a common precursor of dammarane-type saponins, and its sufficient supply is necessary for the efficient synthesis of ginsenoside. RESULTS: In this study, a combinational strategy was used for the construction of an efficient yeast cell factory for PPD production. Firstly, a PPD-producing strain was successfully constructed by modular engineering in Saccharomyces cerevisiae BY4742 at the multi-copy sites. Then, the INO2 gene, encoding a transcriptional activator of the phospholipid biosynthesis, was fine-tuned to promote the endoplasmic reticulum (ER) proliferation and improve the catalytic efficiency of ER-localized enzymes. To increase the metabolic flux of PPD, dynamic control, based on a carbon-source regulated promoter PHXT1, was introduced to repress the competition of sterols. Furthermore, the global transcription factor UPC2-1 was introduced to sterol homeostasis and up-regulate the MVA pathway, and the resulting strain BY-V achieved a PPD production of 78.13 ± 0.38 mg/g DCW (563.60 ± 1.65 mg/L). Finally, sugarcane molasses was used as an inexpensive substrate for the first time in PPD synthesis. The PPD titers reached 1.55 ± 0.02 and 15.88 ± 0.65 g/L in shake flasks and a 5-L bioreactor, respectively. To the best of our knowledge, these results were new records on PPD production. CONCLUSION: The high-level of PPD production in this study and the successful comprehensive utilization of low-cost carbon source -sugarcane molassesindicate that the constructed yeast cell factory is an excellent candidate strain for the production of high-value-added PPD and its derivativeswith great industrial potential.

The Prebiotic Activity of Simulated Gastric and Intestinal Digesta of Polysaccharides from the Hericium erinaceus.

Hericium erinaceus (HE) is a well-known edible and medicinal fungus widely grown in Asian countries. Polysaccharides from the Hericium erinaceus (HEP) are major biological macromolecules. It has been reported that HEP has multiple biological activities, such as antioxidant activity, immunomodulatory effects, anti-inflammatory effect, anti-chronic gastritis activity, and so on. In the current study, we investigated the biological property of HEP during gastrointestinal digestion. The results indicated that both simulated gastric and small intestinal digesta of HEP has better stimulation of probiotics growth than HEP alone, especially for Lactobacillus plantarum BG112. The prebiotic activity was the strongest when HEP was treated by simulated gastric juice for 2 h and by simulated small intestinal juice for 4 h. The molecular weight (Mw) of HEP decreased from 1.68 × 106 Da and 2.32 × 104 Da to 529.3 ± 7.2 Da, as digestion time increased. Meanwhile, the reducing sugar content was significantly increased from 0.610 ± 0.007 to 22.698 ± 0.752 mg/ml, suggesting that the decrease of Mw was likely due to the breakdown of glycosidic bonds. Considerable mannose and galactopyranose were released throughout the gastrointestinal digestion period, indicating that the gastrointestinal digestion resulted in production of free monosaccharides. After fermentation of L. plantarum BG112, the Mw of HEP was decreased and short chain fatty acids (SCFAs) including acetic acid, isovaleric acid, lactic acid, and butyric acid were produced. We speculated that the release of free monosaccharides during gastrointestinal digestion and utilization of HEP, by the probiotics, contributed to the prebiotic activity of HEP's gastric and intestinal digesta. These results unveiled some mechanisms on the close relationship between the structure and bioactivity of polysaccharides, during digestion.

Production and pharmaceutical research of minor saponins in Panax notoginseng (Sanqi): Current status and future prospects.

Panax notoginseng (Burk.) F.H. Chen is a traditional medicinal herb known as Sanqi or Tianqi in Asia and is commonly used worldwide. It is one of the main raw ingredients of Yunnan Baiyao, Fu fang dan shen di wan, and San qi shang yao pian. It is also a source of cardiotonic pill used to treat cardiovascular diseases in China, Korea, and Russia. Approximately 270 Panax notoginseng saponins have been isolated and identified as the major active components. Although the absorption and bioavailability of saponins are predominantly dependent on the gastrointestinal biotransformation capacity of an individual, minor saponins are better absorbed into the bloodstream and act as active substances than major saponins. Notably, minor saponins are absent or are present in minimal quantities under natural conditions. In this review, we focus on the strategies for the enrichment and production of minor saponins in P. notoginseng using physical, chemical, enzyme catalytic, and microbial methods. Moreover, pharmacological studies on minor saponins derived from P. notoginseng over the last decade are discussed. This review serves as a meaningful resource and guide, offering scholarly references for delving deeper into the exploration of the minor saponins in P. notoginseng.

Effect of Ganoderma lucidum water extract on flavor volatiles and quality characteristics of set-type yogurt.

This study investigated the effects of several concentrations of Ganoderma lucidum water extract (GLWE) (0, 0.5, 1.0, and 1.5 %) on set-type yogurt's flavor volatiles as well as the physicochemical, textural, and antioxidant activities of the yogurt during storage. The HS-SPME-GC-MS investigation found that adding GLWE increased the amount of flavor volatiles in yogurt, which improved the quality of flavor volatiles produced by the yogurt. The yogurt's water holding capacity, syneresis, color, and texture all showed optimum values at a 0.5 % GLWE concentration, and its total number of flavor volatiles reached 43. To sum up, the incorporation of GLWE at a 0.5 % concentration promotes the development of superior flavor volatiles in yogurt and enhances its qualitative attributes in contrast to the control group.

Hydroxyl group-induced enhancement of antioxidant activity of resveratrol over pterostilbene by binding to lactoferrin.

The reduced antioxidant capacity of trans-resveratrol (Res) than the second generation of Res, namely pterostilbene (Pte), severely prohibits its in-depth intriguing radical-scavenging applications in food formulations. Herein, a unique chemical structure-dependent strategy was proposed to specifically enhance the radical scavenging activity of Res over Pte, relying on the two more hydroxyl groups on the A-benzene ring of Res, thus facilitating its binding with lactoferrin (LF) to form stable complexes through more hydrogen bonds. We prepared LF-Res and LF-Pte complexes, revealed their binding mechanisms by multispectral analysis and molecular docking/dynamics simulations, further evaluated their antioxidant properties via ABTS and DPPH assays and a model of inhibiting apple browning, eventually elucidated their structure-binding-property relationships. This contribution offers a new approach to restore the antioxidant capability of Res, also paves the way to precisely regulate the fascinating bioactivities of hydrophobic compounds by protein-binding in a chemical structure-, especially hydroxyl group-dependent manner.

Comparison of interactions between alpha-lactalbumin and three protopanaxadiol ginsenosides: Impacts on the structure and antitumor properties.

In this research, interactions between α-lactalbumin (ALA) and three protopanaxadiol ginsenosides [20(S)-Rg3, 20(S)-Rh2, and 20(S)-PPD] were compared to explore the effects of similar ligand on structure and cytotoxicity of ALA. Multi-spectroscopy revealed the binding between ALA and ginsenoside changed the conformation of ALA, which related to different structures and solubility of ligands. Scanning electron microscope illustrated that all ALA-ginsenoside complexes exhibited denser structures via hydrophobic interactions. Additionally, the cytotoxic experiments confirmed that the cytotoxicity of ginsenoside was enhanced after binding with ALA. Molecular docking showed all three ginsenosides were bound to the sulcus depression region of ALA via hydrogen bonding and hydrophobic interaction. Furthermore, molecular dynamics simulation elucidated the precise binding sites and pertinent system properties. Among all three composite systems, 20(S)-Rh2 had optimal binding affinity. These findings enhanced understanding of the synergistic utilization of ALA and ginsenosides as functional ingredients in food, medicine, and cosmetics.

Metabolic Engineering of Escherichia coli for High-Level Production of (R)-Acetoin from Low-Cost Raw Materials.

Acetoin is an important four-carbon platform chemical with versatile applications. Optically pure (R)-acetoin is more valuable than the racemate as it can be applied in the asymmetric synthesis of optically active α-hydroxy ketone derivatives, pharmaceuticals, and liquid crystal composites. As a cytotoxic solvent, acetoin at high concentrations severely limits culture performance and impedes the acetoin yield of cell factories. In this study, putative genes that may improve the resistance to acetoin for Escherichia coli were screened. To obtain a high-producing strain, the identified acetoin-resistance gene was overexpressed, and the synthetic pathway of (R)-acetoin was strengthened by optimizing the copy number of the key genes. The engineered E. coli strain GXASR-49RSF produced 81.62 g/L (R)-acetoin with an enantiomeric purity of 96.5% in the fed-batch fermentation using non-food raw materials in a 3-L fermenter. Combining the systematic approach developed in this study with the use of low-cost feedstock showed great potential for (R)-acetoin production via this cost-effective biotechnological process.

Probing the Biotransformation Process of Sclareol by Resting Cells of Hyphozyma roseonigra.

Sclareol glycol is a key starting material with significant market interest for synthesizing high-value ambroxide, a sustainable substitute for ambergris in high-end fragrances. Sclareol glycol can be obtained by biotransformation of sclareol, a labdane-type diterpene, using Hyphozyma roseonigra. However, the pathway and mechanism of sclareol glycol biosynthesis remain unclear. In this study, the dynamic time course of sclareol biotransformation was explored by resting cell assays and several intermediates produced during biotransformation were detected. The results show that (1) sclareol glycol and sclareolide are not interconverted and are potentially synthesized via different metabolic pathways and (2) several putative intermediates resulting from biotransformation are featured with a labdane carbon backbone, including isomerized and oxidized analogues. A plausible transformation pathway of sclareol in H. roseonigra was proposed based on detected metabolites. This study sheds light on the biosynthetic mechanism of sclareol glycol and paves a way for the future biotechnological production of this promising compound.

Complexation of ellagic acid with α-lactalbumin and its antioxidant property.

Ellagic acid possesses numerous bioactivities such as antioxidant activity and anti-inflammatory effect. In this work, the binding interaction between ellagic acid and α-lactalbumin was investigated by multi-spectroscopy and the results suggested that ellagic acid could change the conformation of α-lactalbumin. Chromatographic analysis proved the interaction of α-lactalbumin with ellagic acid taken place in less than 30 min and this interaction was stable. Computer simulations showed that both aromatic clusters Ⅰ and Ⅱ of α-lactalbumin were active sites for ellagic acid. Interestingly, both the results of molecular docking and molecular dynamics simulations suggested that ellagic acid tended to bind to aromatic cluster Ⅱ rather than aromatic cluster Ⅰ. Moreover, α-lactalbumin could enhance the antioxidant property of ellagic acid, indicating that the solubility of ellagic acid might be improved by combining α-lactalbumin. Overall, this work suggested that α-lactalbumin exhibited binding affinity for ellagic acid and enhanced its antioxidant property.

Characterization of a Group of UDP-Glycosyltransferases Involved in the Biosynthesis of Triterpenoid Saponins of Panax notoginseng.

UDP-glycosyltransferase (UGT)-mediated glycosylation is a common modification in triterpene saponins, which exhibit a wide range of bioactivities and important pharmacological effects. However, few UGTs involved in saponin biosynthesis have been identified, limiting the biosynthesis of saponins. In this study, an efficient heterologous expression system was established for evaluating the UGT-mediated glycosylation process of triterpene saponins. Six UGTs (UGTPn17, UGTPn42, UGTPn35, UGTPn87, UGTPn19, and UGTPn12) from Panax notoginseng were predicted and found to be responsible for efficient and direct enzymatic biotransformation of 21 triterpenoid saponins via 26 various glycosylation reactions. Among them, UGTPn87 exhibited promiscuous sugar-donor specificity of UDP-glucose (UDP-Glc) and UDP-xylose (UDP-Xyl) by catalyzing the elongation of the second sugar chain at the C3 or/and C20 sites of protopanaxadiol-type saponins with a UDP-Glc or UDP-Xyl donor, as well as at the C20 site of protopanaxadiol-type saponins with a UDP-Glc donor. Two new saponins, Fd-Xyl and Fe-Xyl, were generated by catalyzing the C3-O-Glc xylosylations of notoginsenoside Fd and notoginsenoside Fe when incubated with UGTPn87. Moreover, the complete biosynthetic pathways of 17 saponins were elucidated, among which notoginsenoside L, vinaginsenoside R16, gypenoside LXXV, and gypenoside XVII were revealed in Panax for the first time. A yeast cell factory was constructed with a yield of Rh2 at 354.69 mg/L and a glycosylation ratio of 60.40% in flasks. Our results reveal the biosynthetic pathway of a group of saponins in P. notoginseng and provide a theoretical basis for producing rare and valuable saponins, promoting their industrial application in medicine and functional foods.

Characterization and antibacterial activity study of α-Lactalbumin-carvacrol complex.

Food contamination and poisoning caused by bacteria will endanger human health, and the development of natural antibacterial agents is a pressing issue. We prepared ALA-Car complex and demonstrated its formation by multi-spectroscopy techniques and localized surface plasmon resonance experiments. Computer simulations have shown that van der Waals forces dominate the interaction between ALA and Car. The minimum inhibitory concentration (MIC) of Car toward Gram-negative Escherichia coli was decreased from 336 µg/mL to 224 µg/mL after binding to ALA. It had little effect on the MIC of Gram-positive Staphylococcus aureus (224 µg/mL), but further proved Car had a weaker antibacterial activity than the ALA-Car complex by the spread plate method. Overall, this work demonstrated that the ALA-Car complex had significantly higher antibacterial activities than Car, further advancing the development of natural antibacterial agents.

Characterization of resistant starch nanoparticles prepared via debranching and nanoprecipitation.

Cyperus esculentus starch was treated by pullulanase debranching and nanoprecipitation to prepare resistant starch nanoparticles. Amylose contents, rheological properties of debranched starch and the size, crystalline structure, resistant starch contents of the prepared starch nanoparticles were investigated. The results of amylose contents showed that enzymatic hydrolysis 4 h was the most appropriate enzymatic hydrolysis time. Dynamic light scattering analysis and scanning electron microscopy observations showed that when the starch solution was added to the ethanol, the larger the amount of ethanol, the more conducive to the formation of small size starch nanoparticles. When volume ratio of starch solution/ethanol was 1/5, the particle size was 271.1 nm, the content of resistant starch was higher (15.28%). X-ray diffraction results indicated that resistant starch nanoparticles had V-type crystalline structure. Pullulanase debranching and nanoprecipitation can be utilized to prepare smaller size of Cyperus esculentus resistant starch with higher efficiency.

Formation and properties of starch-palmitic acid complex nanoparticles and their influence on Pickering emulsions.

The starch-palmitic acid complex nanoparticles were prepared by Cyperus esculentus starch with enzymatic hydrolysis for different times and then complexed with palmitic acid. The FACE and 13C CP/MAS NMR analysis showed that there were more amylose molecules formed and complexed with palmitic acid when starch was treated by enzymatic hydrolysis for 4 h. With the enzymatic hydrolysis time increasing from 0 h to 4 h, the mean size of starch-palmitic acid complex nanoparticles increased from 500 ± 38.83 nm to 567.2 ± 22.32 nm, the size distribution became more uniform, and the crystallinity increased from 14.99% to 47.72%. The starch-palmitic acid complex nanoparticles could be used as a kind of stabilizers to stabilize Pickering emulsions. Rheological properties and storage stability of Pickering emulsions indicted that starch-palmitic acid complex nanoparticles can better stabilize. The starch-palmitic acid complex nanoparticles could be used as stabilizer of Pickering emulsion and encapsulation of bioactive compounds.

Characterization of interactions between whey protein isolate and hyaluronic acid in aqueous solution: Effects of pH and mixing ratio.

Interactions between whey protein isolate (WPI) and hyaluronic acid (HA) were characterized as functions of pH (6.0-1.0) and protein to polysaccharide ratio (R, 1:4-10:1). Intramolecular soluble complexes formed at pHc of 5.6-5.8, followed by intermolecular insoluble complexes formed at pHΦ1 of 4.4-4.6. Complexes at ratios below 4:1 reached maximum optical value at pH 2.4 while samples above 4:1 peaked at pH 3-3.4 then precipitated. WPI/HA coacervates completely dissociated into soluble complex at pH 1.6-1.8 (pHΦ2). WPI/HA mixtures showed shear thinning behavior and elastic property. Whey protein underwent significant α-helix structure change when interacting with HA in range of pHΦ1>pH > pHΦ2 and at low R values (1:4 and 1:2). Scanning electronic microscope (SEM) pictures showed pH and mixing ratio dependent microstructural changes corresponding with phase transition. Data may provide helpful information for further application of WPI/HA complexes in medical, food and cosmetic fields.

Enhanced cytotoxicity and antioxidant capacity of kaempferol complexed with α-lactalbumin.

As a dietary polyphenol, kaempferol exhibits numerous biological activities such as antioxidant and anticancer properties. However, its application is limited because of its poor solubility and low permeability. This work aims to investigate the interaction of kaempferol with α-lactalbumin. Multiple-spectroscopic techniques were used to prove the interaction between kaempferol and α-lactalbumin. UV-vis absorption spectra suggested that the conformation of α-lactalbumin could be changed via binding with kaempferol. The fluorescence quenching test showed that kaempferol significantly quenched the intrinsic fluorescence of α-lactalbumin. Circular dichroism spectroscopy showed that the percent helicity of α-lactalbumin secondary structure increased when combined with kaempferol. In addition, the α-lactalbumin-kaempferol complex showed stronger inhibition ability on the growth of HeLa cells compared with kaempferol alone. The complex also showed higher antioxidant capacity than kaempferol alone. Molecular docking provided three predicted binding sites of α-lactalbumin for kaempferol, as well as five predicted binding poses of kaempferol. The weak intermolecular interactions were the main forces to stabilize the α-lactalbumin-kaempferol complex. Besides, the binding stability between α-lactalbumin and kaempferol was explored by molecular dynamics simulation. In conclusion, this work provides a basis for the potential application of α-lactalbumin as a delivery carrier for kaempferol owing to its nontoxic and biocompatible properties.

A Green Route for High-Yield Production of Tetramethylpyrazine From Non-Food Raw Materials.

2,3,5,6-Tetramethylpyrazine (TMP) is an active pharmaceutical ingredient originally isolated from Ligusticum wallichii for curing cardiovascular and cerebrovascular diseases and is widely used as a popular flavoring additive in the food industry. Hence, there is a great interest in developing new strategies to produce this high-value compound in an ecological and economical way. Herein, a cost-competitive combinational approach was proposed to accomplish green and high-efficiency production of TMP. First, microbial cell factories were constructed to produce acetoin (3-hydroxy-2-butanone, AC), an endogenous precursor of TMP, by introducing a biosynthesis pathway coupled with an intracellular NAD+ regeneration system to the wild-type Escherichia coli. To further improve the production of (R)-AC, the metabolic pathways of by-products were impaired or blocked stepwise by gene manipulation, resulting in 40.84 g/L (R)-AC with a high optical purity of 99.42% in shake flasks. Thereafter, an optimal strain designated GXASR11 was used to convert the hydrolysates of inexpensive feedstocks into (R)-AC and achieved a titer of 86.04 g/L within 48 h in a 5-L fermenter under optimized fermentation conditions. To the best of our knowledge, this is the highest (R)-AC production with high optical purity (≥98%) produced from non-food raw materials using recombinant E. coli. The supernatant of fermentation broth was mixed with diammonium phosphate (DAP) to make a total volume of 20 ml and transferred to a high-pressure microreactor. Finally, 56.72 g/L TMP was obtained in 3 h via the condensation reaction with a high conversion rate (85.30%) under optimal reaction conditions. These results demonstrated a green and sustainable approach to efficiently produce high-valued TMP, which realized value addition of low-cost renewables.

[Construction of squalene producing cell factories and screening, cloning and expression of key genes].

Squalene is widely used in pharmaceutical, nutraceutical, cosmetics and other fields because of its strong antioxidative, antibacterial and anti-tumor activities. In order to produce squalene, a gene ispA encoding farnesyl pyrophosphate synthase was overexpressed in a previously engineered Escherichia coli strain capable of efficiently producing terpenoids, resulting in a chassis strain that efficiently synthesizes triterpenoids. Through phylogenetic analysis, screening, cloning and expression of squalene synthase derived from different prokaryotes, engineered E. coli strains capable of efficiently producing squalene were obtained. Among them, squalene produced by strains harboring squalene synthase derived from Thermosynechococcus elongatus and Synechococcus lividus reached (16.5±1.4) mg/g DCW ((167.1±14.3) mg/L broth) and (12.0±1.9) mg/g DCW ((121.8±19.5) mg/L broth), respectively. Compared with the first-generation strains harboring the human-derived squalene synthase, the squalene synthase derived from T. elongatus and S. lividus remarkably increased the squalene production by 3.3 times and 2.4 times, respectively, making progress toward the cost-effective heterologous production of squalene.