With staggering progress on genetic manipulation strategies, Saccharomyces cerevisiae is becoming an ideal cell factory for the de novo biosynthesis of lipid compounds. However, due to their hydrophobicity, lipids tend to be accumulated within intracellular spaces and cause a high burden on cell activity and induce product inhibition effect, which ultimately restricted the lipids biomanufacturing for industrial application. Herein, an oleic acid stress (OAS) model was applied for the long-time domestication of BY4741 cells, and a subclone of A-22 was obtained through a series of acclimation (0.1% glucose and 0.2% oleic acid), showing increased accumulation of both biomass and intracellular lipid droplets compared to WT. Comparative transcriptome analysis indicated that compared to fatty acid metabolism, most transcripts enriched in the pathways of glucose catabolism (glycolysis and citrate cycle) and lipid synthesis (phospholipid and sterol) were down-regulated under OAS. While interestingly, most the above transcripts tended to be ‘restored’ in adapted strain A-22. In addition, for physical adaptation, significant increase of phosphatidylcholines was identified by lipidomic analysis, which probably caused the subsequent subcellular expansion of peroxisomes and lipid droplets as observed in the adapted strain, since phosphatidylcholines are the major constituent of their membranes. The present study systematically investigated both the phenotype change and molecular mechanism on adaptation of S. cerevisiae towards oily environment. Detailed information on functional transcripts may provide novel rational modification targets to reinforce the hydrophobic lipids biosynthesis within S. cerevisiae engineered cell factory.
Cronobacter sakazakii, an emerging opportunistic pathogen, is implicated in severe foodborne outbreak infections in premature and full-term infants. Generally, acid tolerance is vital for the pathogenesis of foodborne pathogens; however, its role in C. sakazakii virulence remains largely unknown. To screen out acid-tolerance determinants from transposon mutants, anovel counterselection method using gentamicin and acid was developed. Using the counterselection method and growth assay, we screened several acid-sensitive mutants and found that nlpD encodes an acid-resistance factor in C. sakazakii. Compared to the wild-type strain, the nlpD mutant exhibited attenuated virulence in a rat model. Using macrophage THP-1 cells and a pH probe, we verified that nlpD enables bacteria to resist macrophages by resisting acidification. Finally, we confirmed that nlpD maintains C. sakazakii membrane integrity in acid using propidium iodide permeabilization assays via flow cytometry. Our results confirm that nlpD is a novel virulence factor that permits C. sakazakii to survive under acid stress conditions. Considering that NlpD is a conserved lipoprotein located in the bacterial outer membrane, NlpD could be used as a target for drug development.
Liu Dehong,Shi Hongjie,Chen Guanglin,Zhang Xu,Gu Tingyue,Zhu Minglong,Tan Wensong
High metal ion concentrations and low pH cause severely inhibit the activity of an acidophilic microbial consortium (AMC) in bioleaching. This work investigated the effects of exogenous spermine on biofilm formation and the bioleaching efficiency of LiCoO 2 by AMC in 9K medium. After the addition of 1 mM spermine, the activities of glutathione peroxidase and catalase increased, while the amount of H 2 O 2 , intracellular reactive oxygen species (ROS) and malondialdehyde in AMC decreased. These results indicated that the ability of AMC biofilm to resist oxidative stress introduced by 3.5 g/L Li + and 30.1 g/L Co 2+ was improved by spermine. The activity of glutamate decarboxylase was promoted to restore the intracellular pH buffering ability of AMC. Electrochemical measurements showed that the oxidation rate of pyrite was increased by exogenous spermine. As a result, high bioleaching efficiencies of 97.1% for Li + and 96.1% for Co 2+ from a 5.0% (w v −1 ) lithium cobalt oxide powder slurry were achieved. This work demonstrated that Tafel polarization can be used to monitor the AMC biofilm’s ability of uptaking electrons from pyrite during bioleaching. The corrosion current density increased with 1 mM spermine, indicating enhanced electron uptake by the biofilm from pyrite.
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