Engineering an Artificial Membrane Vesicle Trafficking System (AMVTS) for the Excretion of β-Carotene in Escherichia coli

Large hydrophobic molecules, such as carotenoids, cannot be effectively excreted from cells by natural transportation systems. These products accumulate inside the cells and affect normal cellular physiological functions, which hinders further improvement of carotenoid production by microbial cell factories. In this study, we proposed to construct a novel artificial transport system utilizing membrane lipids to carry and transport hydrophobic molecules. Membrane lipids allow the physiological mechanism of membrane dispersion to be reconstructed and amplified to establish a novel artificial membrane vesicle transport system (AMVTS). Specifically, a few proteins in E. coli were reported or proposed to be related to the formation mechanism of outer membrane vesicles, and were individually knocked out or overexpressed to test their physiological functions. The effects on tolR and nlpI were the most significant. Knocking out both tolR and nlpI resulted in a 13.7% increase of secreted β-carotene with a 35.6% increase of specific production. To supplement the loss of membrane components of the cells due to the increased membrane vesicle dispersion, the synthesis pathway of phosphatidylethanolamine was engineered. While overexpression of AccABCD and PlsBC in TW-013 led to 15% and 17% increases of secreted β-carotene, respectively, the overexpression of both had a synergistic effect and caused a 53-fold increase of secreted β-carotene, from 0.2 to 10.7 mg/g dry cell weight (DCW). At the same time, the specific production of β-carotene increased from 6.9 to 21.9 mg/g DCW, a 3.2-fold increase. The AMVTS was also applied to a β-carotene hyperproducing strain, CAR025, which led to a 24-fold increase of secreted β-carotene, from 0.5 to 12.7 mg/g DCW, and a 61% increase of the specific production, from 27.7 to 44.8 mg/g DCW in shake flask fermentation. The AMVTS built in this study establishes a novel artificial transport mechanism different from natural protein-based cellular transport systems, which has great potential to be applied to various cell factories for the excretion of a wide range of hydrophobic compounds.