RESUMO
Acetic acid is a common inhibitor present in lignocellulose hydrolysate, which inhibits the ethanol production by yeast strains. Therefore, the cellulosic ethanol industry requires yeast strains that can tolerate acetic acid stress. Here we demonstrate that overexpressing a yeast native arginase-encoding gene, CAR1, renders Saccharomyces cerevisiae acetic acid tolerance. Specifically, ethanol yield increased by 27.3% in the CAR1-overexpressing strain compared to the control strain under 5.0 g/L acetic acid stress. The global intracellular amino acid level and compositions were further analyzed, and we found that CAR1 overexpression reduced the total amino acid content in response to acetic acid stress. Moreover, the CAR1 overexpressing strain showed increased ATP level and improved cell membrane integrity. Notably, we demonstrated that the effect of CAR1 overexpression was independent of the spermidine and proline metabolism, which indicates novel mechanisms for enhancing yeast stress tolerance. Our studies also suggest that CAR1 is a novel genetic element to be used in synthetic biology of yeast for efficient production of fuel ethanol.
RESUMO
AIM: Most studies on the immune effect of gp96 were focused on its enhancement of CTLs. It is interesting to know whether gp96 could influence the humoral immune response, and whether the recombinant N-terminal fragment of gp96 could substitute native gp96 to stimulate the immune system. METHODS: gp96 isolated from livers of normal mice and its N-terminal fragment (amino acid 22-355) expressed in E coli were used for immunization of BALb/c mice. Eight groups of mice received one of the following regiments subcutaneously in 100 microL phosphate buffered saline (PBS) at an interval of 3 wk. Group 1: PBS only; group 2: gp96 only; group 3: N-terminal fragment only; group 4: HBsAg only; group 5: HBsAg+gp96; group 6: HBsAg+N-terminal fragment; group 7: HBsAg+incomplete Freud's adjuvant; group 8: HBsAg+N-terminal fragment (95 degrees heated for 30 min). Serum anti-HBsAg antibody levels were assayed by ELISA. CTL responses in splenocytes were analyzed by ELISPOT after the last vaccination. RESULTS: The average titer of serum anti-HBsAg antibody in the mice immunized with HBsAg together with gp96 or its N-terminal fragment were much higher than those immunized with HBsAg alone detected by ELISA. The cellular immune response of the mice immunized with HBsAg together with gp96 or its N-terminal fragment was not different with those immunized with HBsAg alone measured by ELISPOT assay. CONCLUSION: gp96 or its N-terminal fragment greatly improved humoral immune response induced by HBsAg, but failed to enhance the CTL response, which demonstrated the potential of using gp96 or its N-terminal fragment as a possible adjuvant to augment humoral immune response against HBV infection.
Assuntos
Antígenos de Neoplasias/imunologia , Antígenos de Superfície da Hepatite B/imunologia , Vacinas contra Hepatite B/imunologia , Hepatite B/prevenção & controle , Animais , Antígenos de Neoplasias/isolamento & purificação , Feminino , Hepatite B/imunologia , Fígado , Camundongos , Camundongos Endogâmicos BALB C , Fragmentos de Peptídeos/imunologia , Fragmentos de Peptídeos/isolamento & purificaçãoRESUMO
Heat shock protein gp96 is a member of HSP90 families. It can elicits both innate and adaptive immune responses. Generally it is essential to obtain enough amounts of pure gp96 to meet the needs of study and application. But the recombinant gp96 in E. coli is easy to degrade and form aggregates in certain conditions. In the experiment, first cloned human gp96 gene into pET-30a vector and expressed the recombinant in E. coli Blstar. Then purified gp96 by Ni-affinity column, anion exchange column and Gel-filtration in turn. In the end, removes most degraded fragments, aggregates and obtains certain amount of soluble gp96, which make an foundation for further investigations of the protein.