Genetic Manipulation of a Lipolytic Yeast Candida aaseri SH14 Using CRISPR-Cas9 System.

A lipolytic yeast Candida aaseri SH14 that can utilise long-chain fatty acids as the sole carbon source was isolated from oil palm compost. To develop this strain as a platform yeast for the production of bio-based chemicals from renewable plant oils, a genetic manipulation system using CRISPR-Cas9 was developed. Episomal vectors for expression of Cas9 and sgRNA were constructed using an autonomously replicating sequence isolated from C. aaseri SH14. This system guaranteed temporal expression of Cas9 for genetic manipulation and rapid curing of the vector from transformed strains. A ß-oxidation mutant was directly constructed by simultaneous disruption of six copies of acyl-CoA oxidases genes (AOX2, AOX4 and AOX5) in diploid cells using a single sgRNA with 70% efficiency and the Cas9 vector was efficiently removed. Blocking of ß-oxidation in the triple AOX mutant was confirmed by the accumulation of dodecanedioic acid from dodecane. Targeted integration of the expression cassette for C. aaseri lipase2 was demonstrated with 60% efficiency using this CRISPR-Cas9 system. This genome engineering tool could accelerate industrial application of C. aaseri SH14 for production of bio-based chemicals from renewable oils.

Urine NMR Metabolomic Study on Biochemical Activities to Investigate the Effect of P. betle Extract on Obese Rats.

The present studies are to evaluate the ability of PB to induce weight loss and urine metabolite profile of Piper betle L. (PB) leaf extracts using metabolomics approach. Dried PB leaves were extracted with ethanol 70% and the studies were performed in different groups of rats fed with high fat (HFD) and normal diet (ND). Then, fed with the PB extract with 100, 300, and 500 mg/kg and two negative control groups given water (WTR). The body weights were monitored and evaluated. Urine was collected and 1H NMR-based metabolomics approach was used to detect the metabolite changes. Results showed that PB-treated group demonstrated inhibition of body weight gain. The trajectory of urine metabolites showed that PB-treated group gave the different distribution from week 12 to 16 compared with the control groups. In 1H NMR metabolomic approach analysis, the urine metabolites gave the best separation in principle component 1 and 3, with 40.0% and 9.56% of the total variation. Shared and unique structures (SUS) plot model showed that higher concentration PB-treated group was characterized by high level of indole-3-acetate, aspartate, methanol, histidine, and creatine, thus caused an increased the metabolic function and maintaining the body weight of the animals treated.

Amperometric Biosensor Based on Zirconium Oxide/Polyethylene Glycol/Tyrosinase Composite Film for the Detection of Phenolic Compounds.

A phenolic biosensor based on a zirconium oxide/polyethylene glycol/tyrosinase composite film for the detection of phenolic compounds has been explored. The formation of the composite film was expected via electrostatic interaction between hexacetyltrimethylammonium bromide (CTAB), polyethylene glycol (PEG), and zirconium oxide nanoparticles casted on screen printed carbon electrode (SPCE). Herein, the electrode was treated by casting hexacetyltrimethylammonium bromide on SPCE to promote a positively charged surface. Later, zirconium oxide was mixed with polyethylene glycol and the mixture was dropped cast onto the positively charged SPCE/CTAB. Tyrosinase was further immobilized onto the modified SPCE. Characterization of the prepared nanocomposite film and the modified SPCE surface was investigated by scanning electron microscopy (SEM), Electrochemical Impedance Spectroscopy (EIS), and Cyclic voltamogram (CV). The developed biosensor exhibits rapid response for less than 10 s. Two linear calibration curves towards phenol in the concentrations ranges of 0.075-10 µM and 10-55 µM with the detection limit of 0.034 µM were obtained. The biosensor shows high sensitivity and good storage stability for at least 30 days.