Combinatorial metabolic engineering has been widely established for the development of efficient microbial cell factories to produce the products of interest by precisely regulating the expression levels of multiple genes simultaneously. Here, we report a novel multifunctional CRISPR system that enables simultaneous gene activation, repression, and editing (CRISPR-ARE) with a single Cas9-VPR protein for combinatorial metabolic engineering applications in Saccharomyces cerevisiae. Via gRNA engineering, we achieved orthogonal transcriptional regulations and genome editing using the nuclease active Cas9-VPR fusion protein, individually or in a combinatorial manner. After establishing a system for stable expression of multiple gRNAs on the same plasmid, we first demonstrated CRISPR-ARE for simultaneous mCherry activation, mVenus repression, and ADE2 disruption in a fluorescence reporter strain. Subsequently, we adopted CRISPR-ARE for simple and fast combinatorial metabolic engineering, which improved the production of α-santalene for 2.66-fold in a single step. Because of its simplicity and modularity, the developed CRISPR-ARE system could be applied for facile multifunctional metabolic engineering of microbial cell factories, particularly for which only a few CRISPR proteins have been characterized.

The present study evaluated the protective effect of the natural compound flavonoids of Rosa roxburghii Tratt (FRT) against γ-radiation-induced apoptosis and inflammation in mouse thymus cells in vivo and in vitro. Thymus cells and mice were exposed to Co γ-ray at a dose of 6 Gy. The radiation treatment induced significant cell apoptosis and inflammation. Radiation increased the expressions of cleaved caspase 3/8-10, AIF, and PARP-1, and FRT could mitigate their activation and inhibit subsequent apoptosis in the thymus both in vitro or in vivo. Irradiation increased the mRNA expression of ICAM-1/VCAM-1, IL-1α/IL-6 and TNF-α/NF-κB. Our results also indicated that FRT alleviated gene expression of some inflammatory factors such as ICAM-1/VCAM-1, TNF-α/NF-κB, but not IL-1α/IL-6. Irradiation increased the protein expression levels of ICAM-1/VCAM-1, IL-1α/IL-6 and TNF-α/NF-Κb, and our results also indicated that FRT alleviated protein level expression of certain inflammatory factors such as ICAM-1, IL-1α/IL-6, TNF-α/NF-κB, but not VCAM-1. Our results suggested that FRT enhanced radioprotection at least partially by regulating caspase 3/8-10, AIF, and PARP-1 to reduce apoptosis and by regulating ICAM-1, IL-1α/IL-6, TNF-α/NF-κB to reduce inflammation.

The introduction of multi-gene metabolic pathways is generally the first step for the construction of microbial cell factories and plays an essential role in metabolic engineering and synthetic biology. Here, we developed a “PCR & Go” system for facile integration and assembly of multi-gene pathways into the chromosome of Saccharomyces cerevisiae. The core component of the “PCR & Go” system was an expression chassis, where eight promoter/terminator pairs were pre-installed into the yeast chromosome and PCR amplified gene fragments could be inserted directly for functional expression. In combination with the CRISPR/Cas9 system and a gRNA plasmid library, the β-carotene (three genes), zeaxanthin (four genes), and astaxanthin (five genes) biosynthetic pathways were integrated and assembled into the yeast genome with an efficiency of ~93, ~85, and 69%, respectively, using PCR amplified gene fragments with ~40 bp homology arms in a single step. Therefore, the “PCR & Go” system can be used for fast construction of yeast cell factories harboring multi-gene pathways with high efficiency and flexibility.

The clogging of a dense stream of particles when passing through an orifice occurs ubiquitously in both natural and industrial fields. Since most of the jamming phenomena lead to the negative effects, studying and preventing jamming is of great importance. There are two typical types of jamming due to different types of driving force: (a) gravity-driven jamming and (b) fluid-driven jamming. Among these two types of jamming, the fluid-driven jamming occurs in fluid-driven particle flows, and the initial solid concentration, the fluid velocity, and the orifice-particle size ratio has been demonstrated to have effects on the occurrence of this jamming. Although the individual influence of the initial solid concentration and orifice-particle size ratio on jamming has been studied, the coupled effects of these two factors on jamming are little known. In addition, the complex effects of the fluid velocity on jamming have not been fully discussed. To address these problems, this work performs a three-dimensional simulation of the fluid-driven jamming using the coupled Computational Fluid Dynamics-Discrete Element Method (CFD-DEM) model. At first, the jamming probability under different initial conditions is studied. The jamming probability is displayed on the solid concentration-orifice size ratio plane to illustrate the coupled effects of these two factors on jamming. The simulation results show that the critical solid concentration, at which the jamming probability increases to 1, increases with the orifice-particle size ratio. This is because an orifice with a larger orifice size ratio has a greater particle discharge capacity, which allows more particles to pass through without jamming. Then, we reveal the influence of fluid velocity over a wide range on the fluid-driven jamming type, jamming probability and shape of the jamming dome. To the author's knowledge, this is the first time that the shape of the jamming dome has * Corresponding author. Tel: +86 130 6571 0028. Liuhe Road, Xihu District, College of Civil Engineering and Architecture,

The sedimentation process of granular materials exists ubiquitously in nature and many fields which involve the solid-liquid separation. This paper employs the coupled computational fluid dynamics and discrete element method (CFD-DEM) to investigate the sedimentation process of non-cohesive particles, including the hindered settling stage and the deposition stage. Firstly, the coupled CFD-DEM model for sedimentation is validated by the hindered settling velocity at different solid volume concentrations of suspension , i.e., = 0.05~0.6. Two typical modes of sedimentation are also presented by the concentration profiles and the equal-concentration lines. Then, the comparisons between mono-and poly-dispersed particle system are detailed. In the sedimentation of the poly-dispersed particle system, the segregation phenomenon is simulated. Furthermore, this segregation effect reduces with the increase of the initial solid concentration of suspension. From the simulations, the contact force between every pair of particles can be obtained, hence we demonstrate the "effective stress principle" from the view of the particle contact force by giving the correspondence between the particle contact force and the "effective stress", which is a critical concept of soil mechanics. We also demonstrate the effective stress principle from the view of the contact force acting on particles.Moreover, the deposition stage can be simulated by CFD-DEM method, therefore the solid concentrations of sediment bed max on different conditions are studied. Based on the simulation results of max and the theory of sedimentation, this paper also discusses a method to calculate the critical time when sedimentation ends of two typical modes of sedimentation.