초록
<P><B>Abstract</B></P> <P>Carotenoids are a class of terpenes of commercial interest that exert important biological functions. While various strategies have been applied to engineer β-carotene production in microbial cell factories, no work has been done to study and improve the storage of hydrophobic terpene products inside the heterologous host cells. Although the membrane is thought to be the cell compartment that accumulates hydrophobic terpenes such as β-carotene, direct evidence is still lacking. In this work, we engineered the membrane of <I>Escherichia coli</I> in both its morphological and biosynthetic aspects, as a means to study and improve its storage capacity for β-carotene. Engineering the membrane morphology by overexpressing membrane-bending proteins resulted in a 28% increase of β-carotene specific producton value, while engineering the membrane synthesis pathway led to a 43% increase. Moreover, the combination of these two strategies had a synergistic effect, which caused a 2.9-fold increase of β-carotene specific production value (from 6.7 to 19.6mg/g DCW). Inward membrane stacks were observed in electron microscopy images of the engineered <I>E. coli</I> cells, which indicated that morphological changes were associated with the increased β-carotene storage capacity. Finally, membrane separation and analysis confirmed that the increased β-carotene was mainly accumulated within the cell membrane. This membrane engineering strategy was also applied to the β-carotene hyperproducing strain CAR025, which led to a 39% increase of the already high β-carotene specific production value (from 31.8 to 44.2mg/g DCW in shake flasks), resulting in one of the highest reported specific production values under comparable culture conditions. The membrane engineering strategy developed in this work opens up a new direction for engineering and improving microbial terpene producers. It is quite possible that a wide range of strains used to produce hydrophobic compounds can be further improved using this novel engineering strategy.</P> <P><B>Highlights</B></P> <P> <UL> <LI> This work is first attempt to engineer cell membranes for accumulation of hydrophobic products. </LI> <LI> A strategy was developed to engineer cell membranes in both morphological and biosynthesis aspects. </LI> <LI> β-Carotene yield of a <I>E. coli</I> cell factory was improved 2.9 fold only by engineering of the membrane. </LI> <LI> The majority of the increased β-carotene was proved to be associated with the membranes in the membrane-modified <I>E. coli</I> cell factory. </LI> </UL> </P>