CRISPR interference-mediated gene regulation in Pseudomonas putida KT2440.

Targeted gene regulation is indispensable for reprogramming a cellular network to modulate a microbial phenotype. Here, we adopted the type II CRISPR interference (CRISPRi) system for simple and efficient regulation of target genes in Pseudomonas putida KT2440. A single CRISPRi plasmid was generated to express a nuclease-deficient Cas9 gene and a designed single guide RNA, under control of l-rhamnose-inducible Prha BAD and the constitutive Biobrick J23119 promoter respectively. Two target genes were selected to probe the CRISPRi-mediated gene regulation: exogenous green fluorescent protein on the multicopy plasmid and endogenous glpR on the P. putida KT2440 chromosome, encoding GlpR, a transcriptional regulator that represses expression of the glpFKRD gene cluster for glycerol utilization. The CRISPRi system successfully repressed the two target genes, as evidenced by a reduction in the fluorescence intensity and the lag phase of P. putida KT2440 cell growth on glycerol. Furthermore, CRISPRi-mediated repression of glpR improved both the cell growth and glycerol utilization, resulting in the enhanced production of mevalonate in an engineered P. putida KT2440 harbouring heterologous genes for the mevalonate pathway. CRISPRi is expected to become a robust tool to reprogram P. putida KT2440 for the development of microbial cell factories producing industrially valuable products.

Leucine zipper-mediated targeting of multi-enzyme cascade reactions to inclusion bodies in Escherichia coli for enhanced production of 1-butanol.

Metabolons in nature have evolved to facilitate more efficient catalysis of multistep reactions through the co-localization of functionally related enzymes to cellular organelles or membrane structures. To mimic the natural metabolon architecture, we present a novel artificial metabolon that was created by targeting multi-enzyme cascade reactions onto inclusion body (IB) in Escherichia coli. The utility of this system was examined by co-localizing four heterologous enzymes of the 1-butanol pathway onto an IB that was formed in E. coli through overexpression of the cellulose binding domain (CBD) of Cellulomonas fimi exoglucanase. To target the 1-butanol pathway enzymes to the CBD IB, we utilized a peptide-peptide interaction between leucine zipper (LZ) peptides. We genetically fused the LZ peptide to the N-termini of four heterologous genes involved in the synthetic 1-butanol pathway, whereas an antiparallel LZ peptide was fused to the CBD gene. The in vivo activity of the CBD IB-based metabolon was examined through the determination of 1-butanol synthesis using E. coli transformed with two plasmids containing the LZ-fused CBD and LZ-fused 1-butanol pathway genes, respectively. In vivo synthesis of 1-butanol using the engineered E. coli yielded 1.98g/L of 1-butanol from glucose, representing a 1.5-fold increase over that obtained from E. coli expressing the LZ-fused 1-butanol pathway genes alone. In an attempt to examine the in vitro 1-butanol productivity, we reconstituted CBD IB-based metabolon using CBD IB and individual enzymes of 1-butanol pathway. The 1-butanol productivity of in vitro reconstituted CBD IB-based metabolon using acetoacetyl-CoA as the starting material was 2.29mg/L/h, 7.9-fold higher than that obtained from metabolon-free enzymes of 1-butanol pathway. Therefore, this novel CBD-based artificial metabolon may prove useful in metabolic engineering both in vivo and in vitro for the efficient production of desired products.

A magnetometer-free indoor human localization based on loosely coupled IMU/UWB fusion.

The magnetic distortions in indoor environment affects the accuracy of yaw angle estimation using magnetometer. Thus, the accuracy of indoor localization based on inertial-magnetic sensors will be affected as well. To address this issue, this paper proposes a magnetometer-free solution for indoor human localization and yaw angle estimation. The proposed algorithm fuses a wearable inertial sensor consisting of MEMS-based accelerometer and gyroscope with a portable ultra-wideband (UWB) localization system in a cascaded two-step filter consisting of a tilt Kalman filter and a localization Kalman filter. By benchmarking against an optical motion capture system, the experimental results show that the proposed algorithm can accurately track position and velocity as well as the yaw angle without using magnetometer.

Adaptive Kalman filter for indoor localization using Bluetooth Low Energy and inertial measurement unit.

This paper proposes a novel indoor localization method using the Bluetooth Low Energy (BLE) and an inertial measurement unit (IMU). The multipath and non-line-of-sight errors from low-power wireless localization systems commonly result in outliers, affecting the positioning accuracy. We address this problem by adaptively weighting the estimates from the IMU and BLE in our proposed cascaded Kalman filter (KF). The positioning accuracy is further improved with the Rauch-Tung-Striebel smoother. The performance of the proposed algorithm is compared against that of the standard KF experimentally. The results show that the proposed algorithm can maintain high accuracy for position tracking the sensor in the presence of the outliers.