Development of a flow cytometry-based plating-free system for strain engineering in industrial fungi.

Recent technical advances regarding filamentous fungi have accelerated the engineering of fungal-based production and benefited basic science. However, challenges still remain and limit the speed of fungal applications. For example, high-throughput technologies tailored to filamentous fungi are not yet commonly available for genetic modification. The currently used fungal genetic manipulations are time-consuming and laborious. Here, we developed a flow cytometry-based plating-free system to directly screen and isolate the transformed protoplasts in industrial fungi Myceliophthora thermophila and Aspergillus niger. This system combines genetic engineering via the 2A peptide and the CRISPR-Cas9 system, strain screening by flow cytometry, and direct sorting of colonies for deep-well-plate incubation and phenotypic analysis while avoiding culturing transformed protoplasts in plates, colony picking, conidiation, and cultivation. As a proof of concept, we successfully applied this system to generate the glucoamylase-hyperproducing strains MtYM6 and AnLM3 in M. thermophila and A. niger, respectively. Notably, the protein secretion level and enzyme activities in MtYM6 were 17.3- and 25.1-fold higher than in the host strain. Overall, these findings suggest that the flow cytometry-based plating-free system can be a convenient and efficient tool for strain engineering in fungal biotechnology. We expect this system to facilitate improvements of filamentous fungal strains for industrial applications. KEY POINTS: • Development of a flow cytometry-based plating-free (FCPF) system is presented. • Application of FCPF system in M. thermophila and A. niger for glucoamylase platform. • Hyper-produced strains MtYM6 and AnLM3 for glucoamylase production are generated.

Genetic mapping of folate QTLs using a segregated population in maize.

As essential B vitamin for humans, folates accumulation in edible parts of crops, such as maize kernels, is of great importance for human health. But its breeding is always limited by the prohibitive cost of folate profiling. The molecular breeding is a more executable and efficient way for folate fortification, but is limited by the molecular knowledge of folate regulation. Here we report the genetic mapping of folate quantitative trait loci (QTLs) using a segregated population crossed by two maize lines, one high in folate (GEMS31) and the other low in folate (DAN3130). Two folate QTLs on chromosome 5 were obtained by the combination of F2 whole-exome sequencing and F3 kernel-folate profiling. These two QTLs had been confirmed by bulk segregant analysis using F6 pooled DNA and F7 kernel-folate profiling, and were overlapped with QTLs identified by another segregated population. These two QTLs contributed 41.6% of phenotypic variation of 5-formyltetrahydrofolate, the most abundant storage form among folate derivatives in dry maize grains, in the GEMS31×DAN3130 population. Their fine mapping and functional analysis will reveal details of folate metabolism, and provide a basis for marker-assisted breeding aimed at the enrichment of folates in maize kernels.

Genome-wide identification and transcriptional analysis of folate metabolism-related genes in maize kernels.

BACKGROUND: Maize is a major staple food crop globally and contains various concentrations of vitamins. Folates are essential water-soluble B-vitamins that play an important role as one-carbon (C1) donors and acceptors in organisms. To gain an understanding of folate metabolism in maize, we performed an intensive in silico analysis to screen for genes involved in folate metabolism using publicly available databases, followed by examination of the transcript expression patterns and profiling of the folate derivatives in the kernels of two maize inbred lines. RESULTS: A total of 36 candidate genes corresponding to 16 folate metabolism-related enzymes were identified. The maize genome contains all the enzymes required for folate and C1 metabolism, characterized by highly conserved functional domains across all the other species investigated. Phylogenetic analysis revealed that these enzymes in maize are conserved throughout evolution and have a high level of similarity with those in sorghum and millet. The LC-MS analyses of two maize inbred lines demonstrated that 5-methyltetrahydrofolate was the major form of folate derivative in young seeds, while 5-formyltetrahydrofolate in mature seeds. Most of the genes involved in folate and C1 metabolism exhibited similar transcriptional expression patterns between these two maize lines, with the highest transcript abundance detected on day after pollination (DAP) 6 and the decreased transcript abundance on DAP 12 and 18. Compared with the seeds on DAP 30, 5-methyltetrahydrofolate was decreased and 5-formyltetrahydrofolate was increased sharply in the mature dry seeds. CONCLUSIONS: The enzymes involved in folate and C1 metabolism are conserved between maize and other plant species. Folate and C1 metabolism is active in young developing maize seeds at transcriptional levels.

The mitochondrial folylpolyglutamate synthetase gene is required for nitrogen utilization during early seedling development in arabidopsis.

Investigations into the biochemical processes and regulatory mechanisms of nitrogen (N) utilization can aid in understanding how N is used efficiently in plants. This report describes a deficiency in N utilization in an Arabidopsis (Arabidopsis thaliana) transfer DNA insertion mutant of the mitochondrial folylpolyglutamate synthetase gene DFC, which catalyzes the conjugation of glutamate residues to the tetrahydrofolate during folate synthesis. The mutant seedlings displayed several metabolic changes that are typical of plant responses to low-N stress, including increased levels of starch and anthocyanin synthesis as well as decreased levels of soluble protein and free amino acid, as compared with those in wild-type seedlings when external N was sufficient. More striking changes were observed when dfc seedlings were grown under N-limited conditions, including shorter primary roots, fewer lateral roots, higher levels of glycine and carbon-N ratios, and lower N content than those in wild-type seedlings. Gene expression studies in mutant seedlings revealed altered transcript levels of several genes involved in folate biosynthesis and N metabolism. The biochemical and metabolic changes also suggested that N assimilation is drastically perturbed due to a loss of DFC function. The observation that elevated CO(2) partly rescued the dfc phenotypes suggests that the alterations in N metabolism in dfc may be mainly due to a defect in photorespiration. These results indicate that DFC is required for N utilization in Arabidopsis and provide new insight into a potential interaction between folate and N metabolism.

In-vitro evaluation of immunomodulatory activity of sulphation-modified total ginsenosides derivative-3.

Background: Ginseng has been used in biomedicine to prevent and treat decreased physical and mental capacities. Total ginsenosides (TG) from ginseng root which have antitumor and immune-enhancing properties, are the principal active components of Panax ginseng, while the sulphation-modified TG derivative-3 (SMTG-d3) was expected to enhance the anticancer activity in conventional medicinal treatments. Methods: The chlorosulphonic acid-pyridine technique, used for the sulfation modification of TG to improve their biological activity, and the infrared spectroscopic characteristics of TG and SMTG-d3 were investigated, and the effects of SMTG-d3 on immunocytes and cytokines relevant to tumor treatment were assessed. The MTT assay was used to assess the effect of TG and SMTG-d3 on the cytotoxicity and T-lymphocytic proliferation against mouse splenocytes. The LDH method was employed to evaluate NK activity induced by TG or SMTG-d3. The production levels of splenocytes-secreted IL-2 and IFN-γ and peritoneal macrophages-secreted TNF-α were determined using mouse ELISA kits. Results and discussion: It showed that the ideal conditions for the sulfation modification of TG: the volume ratio of chlorosulfonic acid to pyridine lower than 1:2.5; controlled amount of chlorosulfonic acid; and a yield of 51.5% SMTG-d3 (2 h, < 45°C). SMTG-d3 showed two characteristic absorption peaks at 1,230 cm-1 and 810 cm-1, indicating the formation of sulfuric acid esters and the presence of sulfuric acid groups. SMTG-d3 exhibited higher antitumor immunological activity than TG by promoting the proliferation of T lymphocytes and the production of IFN-γ and TNF-α, thus enhancing NK cell activity, and reducing cytotoxicity. The findings imply sulfated modification represents an effective method of enhancing the immunomodulatory activities of TG and could be used as the basis for developing new drug target compounds; SMTG-d3 can serve as an antitumor immunomodulator and can be considered an effective and prospective herbal formulation in clinical applications.

Manipulation of an α-glucosidase in the industrial glucoamylase-producing Aspergillus niger strain O1 to decrease non-fermentable sugars production and increase glucoamylase activity.

Dextrose equivalent of glucose from starch hydrolysis is a critical index for starch-hydrolysis industry. Improving glucose yield and decreasing the non]-fermentable sugars which caused by transglycosylation activity of the enzymes during the starch saccharification is an important direction. In this study, we identified two key α-glucosidases responsible for producing non-fermentable sugars in an industrial glucoamylase-producing strain Aspergillus niger O1. The results showed the transglycosylation product panose was decreased by more than 88.0% in agdA/agdB double knock-out strains than strain O1. Additionally, the B-P1 domain of agdB was found accountable as starch hydrolysis activity only, and B-P1 overexpression in ΔAΔB-21 significantly increased glucoamylase activity whereas keeping the glucoamylase cocktail low transglycosylation activity. The total amounts of the transglycosylation products isomaltose and panose were significantly decreased in final strain B-P1-3 by 40.7% and 44.5%, respectively. The application of engineered strains will decrease the cost and add the value of product for starch biorefinery.

Development of Genetic Tools in Glucoamylase-Hyperproducing Industrial Aspergillus niger Strains.

The filamentous fungus Aspergillus niger is widely exploited by the fermentation industry for the production of enzymes, particularly glucoamylase. Although a variety of genetic techniques have been successfully used in wild-type A. niger, the transformation of industrially used strains with few conidia (e.g., A. niger N1) or that are even aconidial (e.g., A. niger O1) remains laborious. Herein, we developed genetic tools, including the protoplast-mediated transformation and Agrobacterium tumefaciens-mediated transformation of the A. niger strains N1 and O1 using green fluorescent protein as a reporter marker. Following the optimization of various factors for protoplast release from mycelium, the protoplast-mediated transformation efficiency reached 89.3% (25/28) for N1 and 82.1% (32/39) for O1. The A. tumefaciens-mediated transformation efficiency was 98.2% (55/56) for N1 and 43.8% (28/64) for O1. We also developed a marker-free CRISPR/Cas9 genome editing system using an AMA1-based plasmid to express the Cas9 protein and sgRNA. Out of 22 transformants, 9 albA deletion mutants were constructed in the A. niger N1 background using the protoplast-mediated transformation method and the marker-free CRISPR/Cas9 system developed here. The genome editing methods improved here will accelerate the elucidation of the mechanism of glucoamylase hyperproduction in these industrial fungi and will contribute to the use of efficient targeted mutation in other industrial strains of A. niger.

Anticoccidial Activity of Qinghao Powder Against Eimeria tenella in Broiler Chickens.

Artemisia annua (AAH) is traditionally used as an anti-malarial, expectorant and antipyretic Chinese medicine. The aim of this study was to explore the therapeutic effect of Qinghao Powder (QHP) on chicken coccidiosis, evaluate the safe dosage of QHP, and provide test basis for clinical medication. High-performance liquid chromatography (HPLC) and thin-layer chromatography (TLC) were used to detect artemisinin in Qinghao Powder (QHP) for quality control. The level of artemisinin in QHP was 81.03 mg/g. A total of 210 chicks (14 days of age) were divided randomly into seven groups: three QHP treatments (0.15, 0.30, and 0.60 g/kg), a toltrazuril control (1.00 mL/L), a sulfachloropyrazine sodium control (SSC, 0.30 g/L), an E. tenella-infected control, and a healthy control group. All the groups were inoculated orally with 7 × 104 E. tenella oocysts except for the healthy control group. After seven days of administration, compared with the infected control group, chicks which were administered QHP, SS, and toltrazuril showed less bloody feces, oocyst output, and cecal lesions, and the protection rates were improved. The maximum rBWG and ACI were found in the SS-medicated group, followed by the groups medicated with 0.60 and 0.30 g/kg QHP. Therefore, a 0.30 g/kg dose level of QHP in the feed was selected as the recommend dose (RD) in the target animal safety test, in which 80 broiler chicks (14 days of age) were randomly divided into four major groups (I-healthy control group; II-1× RD; III-3× RD; IV-6× RD), with each group subdivided into two subgroups (A and B) consisting of 10 chicks each. After 7-day (for sub-group A) or 14-day (for sub-group B) administration, compared with the healthy control, treatment-related changes in BWG, feed conversion ratio (FCR), relative organ weight (ROW) of the liver, WBC counts, and levels of RBC, HGB, ALT, AST, and TBIL were detected in the 3× and 6× RD groups. No differences were noted in necropsy for all doses, and histopathological examinations exhibited no QHP-associated signs of toxicity or abnormalities in the liver or kidney. The findings suggest that QHP at a dose of 0.30 g/kg feed would be appropriate for therapy and intermittent treatment of E. tenella-infected chicks, the dosage in clinical applications should be set according to the recommended dose to ensure animal safety.

Synergistic effects of multiple enzymes from industrial Aspergillus niger strain O1 on starch saccharification.

BACKGROUND: Starch is one of the most important renewable polysaccharides in nature for production of bio-ethanol. The starch saccharification step facilitates the depolymerization of starch to yield glucose for biofuels production. The filamentous fungus Aspergillus niger (A. niger) is the most used microbial cell factory for production of the commercial glucoamylase. However, the role of each component in glucoamylases cocktail of A. niger O1 for starch saccharification remains unclear except glucoamylase. RESULTS: In this study, we identified the key enzymes contributing to the starch saccharification process are glucoamylase, α-amylase and acid α-amylase out of 29 glycoside hydrolases from the 6-day fermentation products of A. niger O1. Through the synergistic study of the multienzymes for the starch saccharification in vitro, we found that increasing the amount of α-amylase by 5-10 times enhanced the efficiency of starch saccharification by 14.2-23.2%. Overexpression of acid α-amylase in strain O1 in vivo increased the total glucoamylase activity of O1 cultures by 15.0%. CONCLUSIONS: Our study clarifies the synergistic effects among the components of glucoamylases cocktail, and provides an effective approach to optimize the profile of saccharifying enzymes of strain O1 for improving the total glucoamylase activity.

Toxicological evaluation of the ultrasonic extract from Dichroae radix in mice and wistar rats.

This study was aimed at evaluating the acute and subchronic toxicity of ultrasonic extract of Dichroae radix (UEDR) in mice and rats. High performance liquid chromatography (HPLC) and thin layer chromatogrephy (TLC) were used to detect ß-dichroine and α-dichroine in UEDR for quality control. The levels of ß-dichroine and α-dichroine in UEDR were 1.46 and 1.53 mg/g, respectively. An oral LD50 of 2.43 g/kg BW was observed in acute toxicity test. After 28-day repeated oral administration, compared with the control group, treatment-related changes in body weight (BW) and body weight gain (BWG), lymphocyte counts and ratios, as well as in the relative organ weights (ROWs) of liver, kidney, lung, and heart, were detected in the middle- and high-dose groups (P < 0.05, P < 0.01), no differences were noted in the serum biochemical parameters and necropsy examinations in both sexes at all doses. Histopathological examinations exhibited UEDR-associated signs of toxicity or abnormalities. After 14 days withdrawal, no statistically significant or toxicologically relevant differences were observed in any of the UEDR-treated groups, and the hispathological lesions in the high-dose group were alleviated. Findings showed that long-course and high-dose of UEDR administration was toxic, and showed dose-dependence, the toxic damage was reversible.

Genome-Wide Epigenetic Regulation of Gene Transcription in Maize Seeds.

BACKGROUND: Epigenetic regulation is well recognized for its importance in gene expression in organisms. DNA methylation, an important epigenetic mark, has received enormous attention in recent years as it's a key player in many biological processes. It remains unclear how DNA methylation contributes to gene transcription regulation in maize seeds. Here, we take advantage of recent technologies to examine the genome-wide association of DNA methylation with transcription of four types of DNA sequences, including protein-coding genes, pseudogenes, transposable elements, and repeats in maize embryo and endosperm, respectively. RESULTS: The methylation in CG, CHG and CHH contexts plays different roles in the control of gene expression. Methylation around the transcription start sites and transcription stop regions of protein-coding genes is negatively correlated, but in gene bodies positively correlated, to gene expression level. The upstream regions of protein-coding genes are enriched with 24-nt siRNAs and contain high levels of CHH methylation, which is correlated to gene expression level. The analysis of sequence content within CG, CHG, or CHH contexts reveals that only CHH methylation is affected by its local sequences, which is different from Arabidopsis. CONCLUSIONS: In summary, we conclude that methylation-regulated transcription varies with the types of DNA sequences, sequence contexts or parts of a specific gene in maize seeds and differs from that in other plant species. Our study helps people better understand from a genome-wide viewpoint that how transcriptional expression is controlled by DNA methylation, one of the important factors influencing transcription, and how the methylation is associated with small RNAs.

Synthesis and biological activities of novel pleuromutilin derivatives with a substituted thiadiazole moiety as potent drug-resistant bacteria inhibitors.

A series of novel pleuromutilin derivatives possessing thiadiazole moieties were synthesized via acylation reactions under mild conditions. The in vitro antibacterial activities of the derivatives against methicillin-resistant Staphylococcus aureus, methicillin-resistant Staphylococcus epidermidis, Escherichia coli, and Streptococcus agalactiae were tested by the agar dilution method and Oxford cup assay. The majority of the tested compounds displayed moderate antibacterial activities. Importantly, the three compounds with amino or tertiary amine groups in their side chains, 11, 13b, and 15c, were the most active antibacterial agents. Docking experiments carried out on the peptidyl transferase center (PTC) of 23S rRNA proved that there is a reasonable direct correlation between the binding free energy (ΔGb, kcal/mol) and the antibacterial activity. Moreover, the pharmacokinetic profiles of 11 and 15c in rat were characterized by moderate clearance and oral bioavailability.

Arabidopsis plastidial folylpolyglutamate synthetase is required for seed reserve accumulation and seedling establishment in darkness.

Interactions among metabolic pathways are important in plant biology. At present, not much is known about how folate metabolism affects other metabolic pathways in plants. Here we report a T-DNA insertion mutant (atdfb-3) of the plastidial folylpolyglutamate synthetase gene (AtDFB) was defective in seed reserves and skotomorphogenesis. Lower carbon (C) and higher nitrogen (N) content in the mutant seeds than that of the wild type were indicative of an altered C and N partitioning capacity. Higher levels of organic acids and sugars were detected in the mutant seeds compared with the wild type. Further analysis revealed that atdfb-3 seeds contained less total amino acids and individual Asn and Glu as well as NO3-. These results indicate significant changes in seed storage in the mutant. Defects in hypocotyl elongation were observed in atdfb-3 in darkness under sufficient NO3- conditions, and further enhanced under NO3- limited conditions. The strong expression of AtDFB in cotyledons and hypocotyl during early developmental stage was consistent with the mutant sensitivity to limited NO3- during a narrow developmental window. Exogenous 5-formyl-tetrahydrofolate completely restored the hypocotyl length in atdfb-3 seedlings with NO3- as the sole N source. Further study demonstrated that folate profiling and N metabolism were perturbed in atdfb-3 etiolated seedlings. The activity of enzymes involved in N reduction and assimilation was altered in atdfb-3. Taken together, these results indicate that AtDFB is required for seed reserves, hypocotyl elongation and N metabolism in darkness, providing novel insights into potential associations of folate metabolism with seed reserve accumulation, N metabolism and hypocotyl development in Arabidopsis.

Efficient antibacterial agents: a review of the synthesis, biological evaluation and mechanism of pleuromutilin derivatives.

Pleuromutilins were discovered as natural-product antibiotics in 1950. The modifications of pleuromutilin lead to the successful development of veterinary medicines such as tiamulin and valnemulin. Retapamulin became the first pleuromutilin approved for use in human skin infections. Recent advances have led to the synthesis of pleuromutilins that combine potent antibacterial activity with favorable pharmaceutical properties, and three new pleuromutilins, BC-3781, BC-3205 and BC-7013, have entered clinical trials. In this review, the key pleuromutilin derivatives, as well as related novel derivatives during 2009-2013, and its antibacterial activities, are presented. Moreover, the antibacterial and resistance mechanism are discussed.

Synthesis and antibacterial evaluation of novel pleuromutilin derivatives.

A series of novel pleuromutilin derivatives possessing thioether moiety has been synthesized via acylation reaction under mild conditions. Their in vitro antibacterial activity against methicillin-resistant Staphylococcus aureus, methicillin-resistant Staphylococcus epidermidis, Escherichia coli, and Streptococcus agalactiae were tested by agar dilution method and Oxford cup assay. Among the 17 compounds screened, 14-O-[(4-methoxybenzamide-2- methylpropane-2-yl) thioacetate] mutilin 4i, 14-O-[(2-aminobenzamide-2-methylpropane-2-yl) thioacetate] mutilin 5a and 14-O-[(4-aminobenzamide-2-methylpropane-2-yl) thioacetate] mutilin 5c were resulted as most active antibacterial agents.

Chemical synthesis and biological activities of novel pleuromutilin derivatives with substituted amino moiety.

Novel pleuromutilin derivatives designed based on the structure of valnemulin were synthesized and evaluated for their in vitro antibacterial activities. These pleuromutilin derivatives with substituted amino moiety exhibited excellent activities against methicillin-resistant Staphylococcus aureus, methicillin-resistant Staphylococcus epidermidis, Escherichia coli, and Streptococcus agalactiae. Compound 5b showed the highest antibacterial activities and even exceeded tiamulin. Moreover, the docking experiments provided information about the binding model between the synthesized compounds and peptidyl transferase center (PTC) of 23S rRNA.

In vivo inhibition of NAS preparation on H9N2 subtype AIV.

NAS preparation, a kind of Chinese herbal medicine found by the Yunnan Eco-agricultural Research Institute, has potential antiviral activity. In this paper, the inhibiting effect of NAS preparation on H9N2 subtype Avian influenza virus (AIV) was investigated in vivo. Chickens infected with H9N2 virus were treated with NAS preparation for 4 days. The virus was then detected by hemoagglutination (HA) test and reverse transcription polymerase chain reaction (RT-PCR). The results showed that no H9N2 virus could be detected at the 7th day when the chickens were treated with 0.2 g/kg/d or 0.1 g/kg/d of NAS preparation. However the virus could be detected in other chickens without NAS preparation treatment. This result suggested that NAS preparation may be a potential drug candidate to control infection of H9N2 subtype AIV in chickens.