Optimizing liquid fermentation for Wolfiporia cocos: gene expression and biosynthesis of pachymic acid and mycelial biomass.

Wolfiporia cocos, a versatile fungus acclaimed for its nutritional and therapeutic benefits in Traditional Chinese Medicine, holds immense potential for pharmaceutical and industrial applications. In this study, we aimed to optimize liquid fermentation techniques and culture medium composition to maximize mycelial biomass (MB) yield, pachymic acid (PA) concentration, and overall PA production. Additionally, we investigated the molecular basis of our findings by quantifying the expression levels of genes associated with PA and MB biosynthesis using quantitative real-time polymerase chain reaction. Under the optimized fermentation conditions, significant results were achieved, with maximum MB reaching 6.68 g l-1, PA content peaking at 1.25 mg g-1, and a total PA yield of 4.76 g l-1. Notably, among the four examined genes, squalene monooxygenase, exhibited enhanced expression at 0.06 ratio under the optimized conditions. Furthermore, within the realm of carbohydrate-active enzymes, the glycoside hydrolases 16 family displayed elevated expression levels at 21 ratios, particularly during MB production. This study enhances understanding of genetic mechanism governing MB and PA production in W. cocos, highlighting the roles of squalene monooxygenase and glycoside hydrolases 16 carbohydrate-active enzymes.

An omics-based characterization of Wolfiporia cocos reveals three CYP450 members involved in the biosynthetic pathway of pachymic acid.

Wolfiporia cocos is a medicinal mushroom used in China. It biosynthesizes pachymic acid (PA), a main therapeutic triterpene associated with therapies. Nowadays, the unknown PA biosynthesis leads to difficulties in increasing its content in W. cocos. Herein, we report sequencing, assembling, and characterization of the genome and several transcriptomes of W. cocos. Sequence mining determined candidate genes that encode lanosterol synthase, sterol O-acyltransferase, and sterol C-24 methyltransferase likely involved in the steps from lanosterol to PA. Gene cluster analysis identified four CYP450 cDNAs likely involved in the biosynthesis of PA, namely WcCYP64-1, WcCYP64-2, WcCYP52, and WcCYP_FUM15, which were subjected to both overexpression and silencing in mycelia. The overexpression of each of WcCYP64-1, WcCYP52 and WcCYP_FUM15 increased the content of PA, 16α-hydroxytrametenolic acid, eburicoic acid, and tumulosic acid, while the silencing of each gene either significantly or slightly decreased the contents of these four compounds, indicating their involvement in the PA biosynthesis. In addition, different temperatures affected the expression of these genes and the formation of PA. By contrast, the overexpression and silencing of WcCYP64-2 did not alter the formation of these compounds. Taken together, these findings determine more potential steps in the biosynthetic pathway of PA for metabolic engineering.

Versatile roles of polyamines in improving abiotic stress tolerance of plants.

In recent years, extreme environmental cues such as abiotic stresses, including frequent droughts with irregular precipitation, salinity, metal contamination, and temperature fluctuations, have been escalating the damage to plants' optimal productivity worldwide. Therefore, yield maintenance under extreme events needs improvement in multiple mechanisms that can minimize the influence of abiotic stresses. Polyamines (PAs) are pivotally necessary for a defensive purpose under adverse abiotic conditions, but their molecular interplay in this remains speculative. The PAs' accretion is one of the most notable metabolic responses of plants under stress challenges. Recent studies reported the beneficial roles of PAs in plant development, including metabolic and physiological processes, unveiling their potential for inducing tolerance against adverse conditions. This review presents an overview of research about the most illustrious and remarkable achievements in strengthening plant tolerance to drought, salt, and temperature stresses by the exogenous application of PAs. The knowledge of underlying processes associated with stress tolerance and PA signaling pathways was also summarized, focusing on up-to-date evidence regarding the metabolic and physiological role of PAs with exogenous applications that protect plants under unfavorable climatic conditions. Conclusively, the literature proposes that PAs impart an imperative role in abiotic stress tolerance in plants. This implies potentially important feedback on PAs and plants' stress tolerance under unfavorable cues.

Genome sequencing of Colletotrichum gloeosporioides ES026 reveals plausible pathway of HupA.

BACKGROUND: Colletotrichum gloeosporioides ES026, isolated as an endophytic fungal strain, was found to produce the important medicinal compound HuperzineA (HupA). In a genetic context, ES026 showed potential in elucidating the biosynthetic pathway of HupA. METHODS AND RESULTS: The ES026 strain was sequenced using de-novo Illumina sequencing methods in this study. Assembling the cleaned data resulted in 58,594,804bp, consisting of 404 scaffolds. The G + C mol % content of this genome was 52.53%. The genome progressive-alignment with other 4 Colletotrichum strains revealed that ES026 showed closer relation with 030206, SMCG1#C and Nara gc5. More than 60 putative biosynthetic clusters were predicted with the fungal version antiSMASH4.0 program. More than 33 types I polyketide-related biosynthetic gene clusters were distributed, containing PKS and PKS-NRPS (polyketide-nonribosomal peptides) hybrid gene clusters. Another 8 NRPS biosynthetic gene clusters were distributed among the genome of ES026. The prenyltransferases, probably involved in aromatic prenyl-compounds and terpenoid biosynthesis, were analyzed using bioinformatics tools like MEGA. CONCLUSION: We predicted a new possible biosynthetic pathway for the HupA from the pipecolic acid, based on the published HupA biosynthesis proposed pathway, the biosynthesis and pipecolic acid-derived compounds. We hypothesize that a hybrid PKS-NRPS mega-enzyme was probably involved in the biosynthesis of HupA with the pipecolic acid, the building block of rapamycin, as a HupA precursor. The rapamycin is produced from a polyketide biosynthesis pathway, and the domain incorporating the pipecolic acid is studied.

Biosynthetic pathways of triterpenoids and strategies to improve their Biosynthetic Efficiency.

"Triterpenoids" can be considered natural products derived from the cyclization of squalene, yielding 3-deoxytriterpenes (hydrocarbons) or 3-hydroxytriterpenes. Triterpenoids are metabolites of these two classes of triterpenes, produced by the functionalization of their carbon skeleton. They can be categorized into different groups based on their structural formula/design. Triterpenoids are an important group of compounds that are widely used in the fields of pharmacology, food, and industrial biotechnology. However, inadequate synthetic methods and insufficient knowledge of the biosynthesis of triterpenoids, such as their structure, enzymatic activity, and the methods used to produce pure and active triterpenoids, are key problems that limit the production of these active metabolites. Here, we summarize the derivatives, pharmaceutical properties, and biosynthetic pathways of triterpenoids and review the enzymes involved in their biosynthetic pathway. Furthermore, we concluded the screening methods, identified the genes involved in the pathways, and highlighted the appropriate strategies used to enhance their biosynthetic production to facilitate the commercial process of triterpenoids through the synthetic biology method.

Rhizosphere microbial diversity in rhizosphere of Pinellia ternata intercropped with maize.

Dry tubers of Pinellia ternata (Thunb.) Breit are used in traditional Chinese medicine. Commonly known as "banxia" in China, the tubers contain valuable compounds, including alkaloids and polysaccharides that are widely used in pharmaceuticals. The quantity and quality of these important compounds are affected by whether P. ternata is grown as a sole crop or as an intercrop, and P. ternata cultivation has become challenging in recent years. By intercropping P. ternata, its maximum yield, as well as large numbers of chemical components, can be realized. Here, a large data set derived from next-generation sequencing was used to compare changes in the bacterial communities in rhizosphere soils of P. ternata and maize grown as sole crops and as intercrops. The overall microbial population in the rhizosphere of intercropped P. ternata was significantly larger than that of sole-cropped P. ternata, whereas the numbers of distinct microbial genera, ranging from 552 to 559 among treatments, were not significantly different between the two rhizospheres. The relative abundances of the genera differed. Specifically, the numbers of Acidobacteria and Anaerolineaceae species were significantly greater, and those of Bacillus were significantly lower, in the intercropped P. ternata rhizosphere than in the sole-cropped rhizosphere. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s13205-021-03011-3.

Expression Profiling of Flavonoid Biosynthesis Genes and Secondary Metabolites Accumulation in Populus under Drought Stress.

Flavonoids are key secondary metabolites that are biologically active and perform diverse functions in plants such as stress defense against abiotic and biotic stress. In addition to its importance, no comprehensive information has been available about the secondary metabolic response of Populus tree, especially the genes that encode key enzymes involved in flavonoid biosynthesis under drought stress. In this study, the quantitative real-time polymerase chain reaction (qRT-PCR) analysis revealed that the expression of flavonoid biosynthesis genes (PtPAL, Pt4-CL, PtCHS, PtFLS-1, PtF3H, PtDFR, and PtANS) gradually increased in the leaves of hybrid poplar (P. tremula × P. alba), corresponding to the drought stress duration. In addition, the activity and capacity of antioxidants have also increased, which is positively correlated with the increment of phenolic, flavonoid, anthocyanin, and carotenoid compounds under drought stress. As the drought stress prolonged, the level of reactive oxygen species such as hydrogen peroxide (H2O2) and singlet oxygen (O2-) too increased. The concentration of phytohormone salicylic acid (SA) also increased significantly in the stressed poplar leaves. Our research concluded that drought stress significantly induced the expression of flavonoid biosynthesis genes in hybrid poplar plants and enhanced the accumulation of phenolic and flavonoid compounds with resilient antioxidant activity.

Endophytic Fungal Community of Huperzia serrata: Diversity and Relevance to the Production of Huperzine A by the Plant Host.

As the population ages globally, there seem to be more people with Alzheimer's disease. Unfortunately, there is currently no specific treatment for the disease. At present, Huperzine A (HupA) is one of the best drugs used for the treatment of Alzheimer's disease and has been used in clinical trials for several years in China. HupA was first separated from Huperzia serrata, a traditional medicinal herb that is used to cure fever, contusions, strains, hematuria, schizophrenia, and snakebite for several hundreds of years in China, and has been confirmed to have acetylcholinesterase inhibitory activity. With the very slow growth of H. serrata, resources are becoming too scarce to meet the need for clinical treatment. Some endophytic fungal strains that produce HupA were isolated from H. serrate in previous studies. In this article, the diversity of the endophytic fungal community within H. serrata was observed and the relevance to the production of HupA by the host plant was further analyzed. A total of 1167 strains were obtained from the leaves of H. serrata followed by the stems (1045) and roots (824). The richness as well as diversity of endophytic fungi within the leaf and stem were higher than in the root. The endophytic fungal community was similar within stems as well as in leaves at all taxonomic levels. The 11 genera (Derxomyces, Lophiostoma, Cyphellophora, Devriesia, Serendipita, Kurtzmanomyces, Mycosphaerella, Conoideocrella, Brevicellicium, Piskurozyma, and Trichomerium) were positively correlated with HupA content. The correlation index of Derxomyces with HupA contents displayed the highest value (CI = 0.92), whereas Trichomerium showed the lowest value (CI = 0.02). Through electrospray ionization mass spectrometry (ESI-MS), it was confirmed that the HS7-1 strain could produce HupA and the total alkaloid concentration was 3.7 ug/g. This study will enable us to screen and isolate the strain that can produce HupA and to figure out the correlation between endophytic fungal diversity with HupA content in different plant organs. This can provide new insights into the screening of strains that can produce HupA more effectively.

Investigating the potential of different jute varieties for phytoremediation of copper-contaminated soil.

Copper (Cu), with many documented cases of Cu toxicity in agriculture lands, is becoming an increasingly common issue in and elsewhere in China. However, fibrous crop such as jute is being used as phytoremediation candidate in Cu-contaminated soils due to its huge biomass. A pot experiment was conducted using four different varieties (HT, C-3, GC, and SH) of jute grown in highly Cu-contaminated soil (2221 mg kg-1), collected from Hubei Province, China. Results from this study showed that C-3 and HT were more resistant to Cu stress, while GC and SH had a serious effect due to high concentration of Cu and a significant decrease in growth and biomass. Furthermore, Cu in roots, leaves, stem core, and bast were higher in C-3 and HT compared with GC and SH. Likewise, at post-harvesting stage, maximum Cu concentration from Cu-contaminated soil was extracted by C-3 and HT while small amount was accumulated by GC and SH. The high content of malondialdehyde (MDA) in the leaves of GC and SH indicated that Cu induced oxidative damage while the antioxidative enzyme activities of superoxidase dismutase (SOD) and peroxidase (POD) were increased to scavenge reactive oxygen species (ROS) formed during oxidative stress in the plants. Conclusively, it can be identified that when grown in Cu-contaminated soil, C-3 and HT have greater ability to grow in polluted soils and possible phytoremediation materials to revoke a large amount of Cu.