Maximizing the flux of farnesyl diphosphate (FPP) to farnesene biosynthesis is the main challenge of farnesene overproduction in Saccharomyces cerevisiae. In this study, we screened α-farnesene synthase from soybean (Fsso) with a higher catalytic ability. Combining the overexpression of the mevalonate (MVA) pathway with the expression of Fsso, an engineered yeast strain producing 190.5 mg/L α-farnesene was screened with poor growth. By decreasing the copies of 3-hydroxy-3-methylglutaryl-coenzyme (HMGR) overexpressed, the titer was increased to 417.8 mg/L. Then, the coexpression of Fsso and HMGR under the control of the GAL promoter and inactivation of lipid phosphate phosphatase encoded by DPP1 promoted the titer to 1163.7 mg/L. The titer was further increased to 1477.2 mg/L at the shake flask level with better growth by the construction of a prototrophic strain. Finally, the highest α-farnesene production of 10.4 g/L in S. cerevisiae was obtained by fed-batch fermentation in a 5 L bioreactor.

The amount of geranylgeranyl diphosphate (GGPP) is vital for microbial production of geranylgeraniol (GGOH) in Saccharomyces cerevisiae. In this study, a GGPP synthase with stronger catalytic ability was used to increase the supply of GGPP, and an engineered strain producing 374.02 mg/L GGOH at the shake flask level was constructed. Then, by increasing the metabolic flux of the mevalonate (MVA) pathway and the supply of isopentenyl pyrophosphate (IPP), the titer was further increased to 772.98 mg/L at the shake flask level, and we achieved the highest GGOH titer to date of 5.07 g/L in a 5 L bioreactor. This is the first report on the utilization of isoprenol for increasing the amount of IPP and enhancing GGOH production in S. cerevisiae. In the future, these strategies and engineered strains can be used to enhance the production of other terpenoids in S. cerevisiae.

The lipase TrLipB from Thermomicrobium roseum is highly thermostable. However, its thermostable skeleton and mechanism of action should be investigated for industrial applications. Toward this, TrLipB was crystallized using the hanging-drop vapor diffusion method and subjected to Xray diffraction at 2.0 Å resolution in this study. The rigid sites, such as the prolines on the relatively flexible loops on the enzyme surface, were scanned. Soft substitutions of these sites were designed using both molecular dynamics (MD) simulation and site-directed mutagenesis. The thermostability of several substitutions decreased markedly, while the catalytic efficiencies of the P9G, P127G, P194G, and P300G mutants reduced substantially; additionally, the thermostable framework of the double mutant, P194G/P300G, was considerably perturbed. However, the substitutions on the lid of the enzyme, including P49G and P48G, promoted the catalytic efficiency to approximately 150% and slightly enhanced the thermostability below 80 °C. In MD simulations, the P100G, P194G, P100G/P194G, P194G/ P300G, and P100G/P194G/P300G mutants showed high B-factors and RMSD values, whereas the secondary structures, radius of gyration, H-bonds, and solvent accessible surface areas of these mutants were markedly affected. Our observations will assist in understanding the natural framework of a stable lipase, which might contribute to its industrial applications.

No abstract

In this study, four kinds of phospholipase A1-metal (Al/Co/Cu/Mn) hybrid nanostructures were prepared for enhancing the stability of the free PLA1. The formed hybrid complexes were characterized by scanning electron microscope (SEM), Fourier infrared spectroscopy (FTIR), and X-ray diffraction (XRD). The stability and substrate specificity of immobilized enzymes were subsequently determined. After immobilization, the temperature tolerance of PLA1–metal hybrid nanostructures was enhanced. The relative activity of PLA1–Al/Co/Cu hybrid nanostructures remained above 60% at 50 °C, while that of free enzyme was below 5%. The thermal transition temperature measured by differential scanning calorimetry (DSC) was found to increase from 65.59 °C (free enzyme) to 173.14 °C, 123.67 °C, 96.31 °C, and 114.79 °C, referring to PLA1–Cu/Co/Al/Mn hybrid nanostructures, respectively. Additionally, after a storage for fourteen days at 4 °C, the immobilized enzymes could exhibit approximately 60% of the initial activity, while the free PLA1 was inactivated after four days of storage. In brief, using Co2+, Cu2+, Al3+, and Mn2+ as the hybridization materials for immobilization could improve the catalytic properties and stability of the free PLA1, suggesting a promising method for a wider application of PLA1 in many fields such as food, cosmetics, and the pharmaceutical industry.