Characterization and optimization of the LAC4 upstream region for low-leakage expression in Kluyveromyces marxianus.

Kluyveromyces marxianus is a promising host for the production of heterologous proteins, chemicals, and bioethanol. One superior feature of this species is its capacity to assimilate lactose, which is rendered by the LAC12-LAC4 gene pair encoding a lactose permease and a ß-galactosidase enzyme. Little is known about the regulation of LAC4 in K. marxianus. In this study, we showed the presence of weak glucose repression in the regulation of LAC4 and that might contribute to the leaky expression of LAC4 in the glucose medium. In a mutagenesis screen of 1000-bp LAC4 upstream region, one mutant region, named H1, drove low-leakage expression of a URA3 reporter gene in glucose medium. Two mutations inside a polyadenosine stretch (poly(A)) of 5' UTR were major contributors to the low-leakage phenotype of H1. H1 directed low-leakage expression of GFP on a plasmid and that of LAC4 in situ in the glucose medium, which was not due to the reduction of mRNA levels. Meanwhile, H1 did not affect the induction of GFP or LAC4 by lactose. Cre recombinase expressed by H1 caused lower toxicity in the repressive condition and achieved higher yield after induction, compared with that expressed by a wild-type LAC4 upstream region or a strong INU1 promoter. Our study suggested that poly(A) inside 5' UTR played a role in regulating the expression of LAC4 in the repressive condition. Meanwhile, H1 provided a base for the development of a strict inducible system for expressing industrial proteins, especially toxic proteins.

Lipid Rafts Promote trans Fatty Acid-Induced Inflammation in Human Umbilical Vein Endothelial Cells.

The effects of two fatty acids, oleic acid (OLA) and elaidic acid (ELA) on normal human umbilical vein endothelial cells (HUVEC) and non-rafts HUVEC were investigated in this study. The expression levels of inflammatory cytokines (ICAM-1, VCAM-1 and IL-6) were analyzed. Western blot was used to analyze the expression levels of inflammation-related proteins (NF-κB, ERK1/2) and toll-like receptors 4 (TLR4). The results showed that the levels of nuclear translocation of NF-κB p65 and phosphorylated ERK1/2 were significantly decreased only in non-lipid rafts cells pretreated with trans fatty acid (TFA). The expression of TLR4 in the ELA-treated normal cells was higher than that in non-lipid rafts HUVEC. When the lipid rafts was destroyed by methyl-ß-cyclodextrin, the levels of nuclear translocation of NF-κB p65, phosphorylated ERK1/2 and TLR4 were decreased significantly. Therefore, lipid rafts may be involved in TFA induced-inflammation in HUVEC through blocking the inflammatory signal pathway. Lipid rafts might be a platform for specific receptors such as TLR4 for TFA to activate the pro-inflammation on cell membranes.