Engineering global transcription to tune lipophilic properties in Yarrowia lipolytica.

BACKGROUND: Evolution of complex phenotypes in cells requires simultaneously tuning expression of large amounts of genes, which can be achieved by reprograming global transcription. Lipophilicity is an important complex trait in oleaginous yeast Yarrowia lipolytica. It is necessary to explore the changes of which genes' expression levels will tune cellular lipophilic properties via the strategy of global transcription engineering. RESULTS: We achieved a strategy of global transcription engineering in Y. lipolytica by modifying the sequences of a key transcriptional factor (TF), SPT15-like (Yl-SPT15). The combinatorial mutagenesis of this gene was achieved by DNA assembly of up to five expression cassettes of its error-prone PCR libraries. A heterologous beta-carotene biosynthetic pathway was constructed to research the effects of combined Yl-SPT15 mutants on carotene and lipid production. As a result, we obtained both an "enhanced" strain with 4.7-fold carotene production and a "weakened" strain with 0.13-fold carotene production relative to the initial strain, nearly 40-fold changing range. Genotype verification, comparative transcriptome analysis, and detection of the amounts of total and free fatty acids were made for the selected strains, indicating effective tuning of cells' lipophilic properties. We exploited the key pathways including RNA polymerase, ketone body metabolism, fatty acid synthesis, and degradation that drastically determined cells' variable lipophilicity. CONCLUSIONS: We have examined the effects of combinatorial mutagenesis of Yl-SPT15 on cells' capacity of producing beta-carotene and lipids. The lipophilic properties in Y. lipolytica could be effectively tuned by simultaneously regulating genome-wide multi-gene expression levels. The exploited gene targets and pathways could guide design and reconstruction of yeast cells for tunable and optimal production of other lipophilic products.

CRISPR/Cas9-based genome editing of the filamentous fungi: the state of the art.

In recent years, a variety of genetic tools have been developed and applied to various filamentous fungi, which are widely applied in agriculture and the food industry. However, the low efficiency of gene targeting has for many years hampered studies on functional genomics in this important group of microorganisms. The emergence of CRISPR/Cas9 genome-editing technology has sparked a revolution in genetic research due to its high efficiency, versatility, and easy operation and opened the door for the discovery and exploitation of many new natural products. Although the application of the CRISPR/Cas9 system in filamentous fungi is still in its infancy compared to its common use in E. coli, yeasts, and mammals, the deep development of this system will certainly drive the exploitation of fungal diversity. In this review, we summarize the research progress on CRISPR/Cas9 systems in filamentous fungi and finally highlight further prospects in this area.

[Comparision of Ecological Risk Assessment Based on the Total Amount and Speciation Distribution of Heavy Metals in Soil:A Case Study for Longyan City, Fujian Province].

A total of 110 topsoil samples and 61 crop samples along the Lantian-Yangdong Villages were collected in Shizhong, Longyan City. The total amount and speciation of heavy metals(Pb, Cd, As) in soil and crops were determined. The characteristics of the absorption of heavy metals by specific crops in the study area were analyzed, and a new method of risk assessment based on the heavy metal speciation and its bioavailability was established by statistical analysis. This new method was used to evaluate the soil ecological risk and to compare it with the traditional method of potential ecological risk index (RI). The results indicated that the Lantian-Yangdong Villages were located in an area where Pb, Cd, and As mainly originate from the natural soil parent material with weak human disturbance. There was no significant Pb or As pollution in the whole region. Cd was the main pollutant with low pollution intensity. Four types of biological components except for the residual form followed the order of Cd(53.28%) > Pb(43.28%) > As(30.71%). Correlation and regression analyses of total metal concentrations, heavy metal speciation, and crop uptake in the study area showed that the correlations between the total amount of heavy metals and the ion exchange state, carbonate state, and other active forms were low; the results even showed nonlinear relationships between those variables.The ion exchange state had the greatest effect on the absorption of Pb, Cd, and As by coix seed and rice. From the perspective of bioavailability, the new method based on the heavy metal speciation was more accurate than the traditional method based on the total amount of heavy metals.

Synthesis and Anti-HIV-1 Activity Evaluation for Novel 3a,6a-Dihydro-1H-pyrrolo[3,4-c]pyrazole-4,6-dione Derivatives.

The search for new molecular constructs that resemble the critical two-metal binding pharmacophore and the halo-substituted phenyl functionality required for HIV-1 integrase (IN) inhibition represents a vibrant area of research within drug discovery. As reported herein, we have modified our recently disclosed 1-[2-(4-fluorophenyl)ethyl]-pyrrole-2,5-dione scaffolds to design 35 novel compounds with improved biological activities against HIV-1. These new compounds show single-digit micromolar antiviral potencies against HIV-1 and low toxicity. Among of them, compound 9g and 15i had potent anti-HIV-1 activities (EC50 < 5 µM) and excellent therapeutic index (TI, CC50/EC50 > 100). These two compounds have potential as lead compounds for further optimization into clinical anti-HIV-1 agents.

Next-generation sequencing-based molecular diagnosis of neonatal hypotonia in Chinese Population.

Neonatal hypotonia is extremely challenging to diagnose because numerous disorders present similar clinical manifestations. Two panels for diagnosing neonatal hypotonia were developed, which enriches 35 genes corresponding to 61 neonatal hypotonia-related disorders. A cohort of 214 neonates with hypotonia was recruited from 2012 to 2014 in China for this study. Of these subjects, twenty-eight neonates with hypotonia were eliminated according to exclusion criteria and 97 were confirmed using traditional detection methods. The clinical diagnoses of the remaining 89 neonates with hypotonia were approached by targeted next-generation sequencing (NGS). Among the 89 tested neonates, 25 potentially pathogenic variants in nine genes (RYR1, MECP2, MUT, CDKL5, MPZ, PMM2, MTM1, LAMA2 and DMPK) were identified in 22 patients. Six of these pathogenic variants were novel. Of the 186 neonates with hypotonia, we identified the genetic causes for 117 neonates by the traditional detection methods and targeted NGS, achieving a high solving rate of 62.9%. In addition, we found seven neonates with RETT syndrome carrying five mutations, thus expanding the mutation profiles in Chinese neonates with hypotonia. Our study highlights the utility of comprehensive molecular genetic testing, which provides the advantage of speed and diagnostic specificity without invasive procedures.

Seed germination, root elongation, root-tip mitosis, and micronucleus induction of five crop plants exposed to chromium in fluvo-aquic soil.

The present study aimed to determine the toxic effects of chromium (Cr) on cabbage (Brassica oleracea), cucumber (Cucumis sativus), lettuce (Lactuca sativa), wheat (Triticum aestivum), and corn (Zea mays), and identify the sensitive plant species and appropriate bioassays for potential use in phytotoxicity assessment of Cr in soil. Results showed that seed germination might not be a sensitive assay for assessing Cr toxicity because at most of the Cr levels there were no toxic effects. Root elongation was more sensitive to Cr than seed germination. The lowest concentration of adverse effect (LOAEC) of lettuce was 20 mg Cr/kg(-1) soil, and that of the other 4 species was 50 mg Cr/kg(-1) soil. The mitotic index fluctuated with increasing Cr concentration, thus it was insufficient to assess toxicity of Cr in soil. However, micronucleus assay showed that 5 mg Cr/kg(-1) soil caused a significant increase in micronucleus frequency in cabbage, cucumber, and lettuce. For wheat and corn, however, the LOAEC was 20 and 50 mg/Cr/kg(-1) soil, respectively. Furthermore, the analysis of Cr accumulation showed that lettuce significantly accumulated Cr for all the tested concentrations. However, corn and wheat significantly accumulated Cr only with the highest tested dose. This may explain the higher inhibitory effects of Cr on root growth. It can be concluded that root elongation and micronucleus assay are good indicators to assess the phytotoxicity of Cr in soil. Lettuce is the most sensitive species for indicating the toxicity of Cr in soil.

The metabolomics of carotenoids in engineered cell factory.

Carotenoids such as beta-carotene, lycopene, and antheraxanthin have plenty of scientific and commercial value. The comprehensive investigation of carotenoids drives people to improve and develop all kinds of analytical techniques to approach or even achieve "versatile" analysis. The metabolic engineering efforts in plants and algae have progressed rapidly, aiming to enable the use of plants and algae as "cell factories" for producing specific or novel carotenoids, such as beta-carotene (provitamin A) in Gold rice, while the emerging technologies of metabolomics support it by providing comprehensive analysis of carotenoids biochemical characterizations. This review describes metabolomics as a high-throughput platform to study carotenoids, including the engineering methods in the plants or algae, the bioinformatics for metabolomics, and the metabolomics of carotenoids in engineered cell factory. Modern systems biology tools, together with the development of genomics and metabolomics databases, will dramatically facilitate the advancement of our knowledge in gene-to-metabolite networks in plants. Metabolomics accompanying genomics, transcriptomics, and proteomics as well as bioinformatics facilitate metabolic engineering efforts towards designing superior biocatalysts in cell factories. Ongoing advances in biological techniques coupled with crucial metabolic networks will further promote plants and algae as attractive platforms for the production of numerous high-value compounds such as carotenoids.