Terminally sialylated N-glycoproteins are of great interest in therapeutic applications. Due to the inability of prokaryotes to carry out this post-translational modification, they are currently predominantly produced in eukaryotic host cells. In this study, we report a synthetic pathway to produce a terminally sialylated N-glycoprotein in the periplasm of Escherichia coli, mimicking the sialylated moiety (Neu5Ac-α-2,6-Gal-β-1,4-GlcNAc-) of human glycans. A sialylated pentasaccharide, Neu5Ac-α-2,6-Gal-β-1,4-GlcNAc-β-1,3-Gal-β-1,3-GlcNAc-, was synthesized through the activity of co-expressed glycosyltransferases LsgCDEF from Haemophilus influenzae, Campylobacter jejuni NeuBCA enzymes, and Photobacterium leiognathi α-2,6sialyltransferase in an engineered E. coli strain which produces CMP-Neu5Ac. C. jejuni oligosaccharyltransferase PglB was used to transfer the terminally sialylated glycan onto a glyco-recognition sequence in the tenth type III cell adhesion module of human fibronectin. Sialylation of the target protein was confirmed by lectin blotting and mass spectrometry. This proof-of-concept study demonstrates the successful production of terminally sialylated, homogeneous N-glycoproteins with α-2,6-linkages in the periplasm of E. coli and will facilitate the construction of E. coli strains capable of producing terminally sialylated N-glycoproteins in high yield.

The direct yaw moment control can effectively enhance the yaw stability of the vehicle under extreme conditions, which has become one of the essential technologies for the distributed driving electric bus. Due to the features of a large mass and high center of gravity of the bus, lateral instability is more likely to occur under extreme driving conditions. To reduce the uncertainty and interference in the yaw movement process of the bus, this paper targets the instability caused by the coupling problem between the sideslip angle and yaw rate. An adaptive fuzzy sliding mode control is proposed to execute direct yaw moment control. The weight coefficient of the sideslip angle and the yaw rate is adjusted via fuzzy control in real time. The optimal direct yaw moment is finally obtained. A distribution method based on the vertical load proportion is adopted for the allocation of four motors’ torque. Under three typical working conditions, a joint simulation test was carried out. The simulation results demonstrate that the raised method decreases the amplitude of the sideslip angle by 20.90%, 12.75%, and 23.67% and the yaw rate is 8.62%, 6.89%, and 9.28%, respectively. The chattering and sudden changes in the additional yaw moment are also lessened. The control strategy can realize the control target, which effectively strengthens the yaw stability of the bus.

This paper suggests a hierarchical coordination control strategy to enhance the stability of distributed drive electric bus. First, an observer based on sliding mode observer (SMO) and adaptive neural fuzzy inference system (ANFIS) was designed to estimate the vehicle state parameters. Then the upper layer of the strategy primarily focuses on coordinating active front steering (AFS) and direct yaw moment control (DYC). The phase plane method is utilized in this layer to provide an assessment basis for the switching control safety of AFS and DYC. The lower layer of the strategy designs an integral terminal sliding mode controller (ITSMC) and a non-singular fast terminal sliding mode controller (NFTSMC) to obtain the optimal additional front wheel steering angle to improve handling performance. A fuzzy sliding mode controller (FSMC) is also proposed to obtain additional yaw moment to ameliorate yaw stability. Finally, the strategy proposed in this paper is subjected to simulation testing and compared with the performance of AFS and DYC systems. The proposed strategy is also evaluated for tracking errors in sideslip angle and yaw rate under two conditions. The results demonstrate that the proposed strategy can effectively adapt to various extreme environments and improve the maneuvering and yaw stability of the bus.

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