Combinatorial metabolic engineering of Saccharomyces cerevisiae for improved production of 7-dehydrocholesterol

7-Dehydrocholesterol(7-DHC),a key pharmaceutical intermediate in the production of vitamin D3,has a wide range of applications.To explore fermentative synthesis of 7-DHC,a 7-DHC-producing Saccharomyces cerevisiae strain was constructed by blocking the competitive pathway,eliminating rate-limiting steps,altering global reg-ulation,and pathway compartmentalization.After blocking the competitive pathway by disrupting ERG5 and ERG6 and introducing DHCR24 from Gallus gallus,S.cerevisiae produced 139.72 mg/L(17.04 mg/g dry cell weight,hereafter abbreviated as DCW)7-DHC.Subsequent alteration of global regulation by deleting ROX1 and overexpressing UPC2-1 increased 7-DHC production to 217.68 mg/L(37.56 mg/g DCW).To remove the accu-mulated squalene,the post-squalene pathway was strengthened by co-overexpression of PGAL1-driven ERG11 and PGAL10-driven ERG1,which improved 7-DHC titer and yield to 281.73 mg/L and 46.78 mg/g DCW,respectively,and reduced squalene content by 90.12%.We surmised that the sterol precursors in the plasma membrane and peroxisomes may not be accessible to the pathway enzymes,thus we re-localized DHCR24p and Erg2p-GGGGS-Erg3p to the plasma membrane and peroxisomes,boosting 7-DHC production to 357.53 mg/L(63.12 mg/g DCW).Iron supplementation further increased 7-DHC production to 370.68 mg/L in shake flasks and 1.56 g/L in fed-batch fermentation.This study demonstrates the power of global regulation and subcellular relocalization of key enzymes to improve 7-DHC synthesis in yeast.

Foraging behavior related to habitat characteristics in the invasive wasp Vespula germanica

When foraging on carrion resources, the wasp Vespula germanica usually makes repeated visits to the feeding site until depleting the resource. In the present study we analyzed how environmental cues affect wasps＇ behavior when re-locating a protein food source. We studied this behavior in two different natural habitats： closed and open habitats. As closed habitats have more references to orient wasps to the feeding site than open habitats, we hypothesized that they would return to the foraging site more frequently in closed habitats than in open ones. We tested this hypothesis by studying wasp behavior in three different natural habitat conditions： （i） closed habitats, （ii） open habitats, and （iii） open habitats artificially modified by adding five sticks with flagging. Experiments consisted of training individual wasps to feed from a certain array, and at the testing phase we removed food and displaced the array by 60 cm. Therefore, we recorded wasps＇ choices when returning to the training area, by counting both the wasps＇ first approaches and the number of visits to the original feeding site and the displaced array. Wasps＇ behavior while re-locating a protein food source was different if foraging at open or closed habitats. Wasps more frequently re- visited a previous feeding location when foraging in closed habitats than when foraging in open ones. Furthermore, wasps more frequently visited the displaced array than the original feeding site in all three treatments. Nevertheless, when wasps were trained in closed habitats, they returned to the original feeding site more frequently than if trained in open ones. Interestingly, when five sticks with flagging were added in open habitats, wasps responded similarly as in closed habitats without these references. The results show that foraging behavior in V. germanica seems to be different in closed and open habitats, probably associated with the existence of references that guide foragers when re-locating undepleted resources