Hexadecane and Tween 80 stimulate lipase production in Burkholderia glumae by different mechanisms

Abstract

Burkholderia glumae strain PG1 produces a lipase of biotechnological relevance. Lipase production by this strain and its derivative LU8093, which was obtained through classical strain improvement, was investigated under different conditions. When 10% hexadecane was included in the growth medium, lipolytic activity in both strains could be increased approximately 7-fold after 24 h of growth. Hexadecane also stimulated lipase production in a strain containing the lipase gene fused to the tac promoter, indicating that hexadecane did not affect lipase gene expression at the transcriptional level, which was confirmed using lipA-gfp reporter constructs. Instead, hexadecane appeared to enhance lipase secretion, since the amounts of lipase in the culture supernatant increased in the presence of hexadecane, with a concomitant decrease in the cells, even when protein synthesis was inhibited with chloramphenicol. In the presence of olive oil as a carbon source, nonionic detergents, such as Tween 80, increased extracellular lipase activity twofold. Like hexadecane, Tween 80 appeared to stimulate lipase secretion, although in a more disruptive manner, since other, normally nonsecreted proteins were found in the culture supernatant. Additionally, like olive oil, Tween 80 was found to induce lipase gene expression in strain PG1 in medium containing sucrose as a carbon source but not in glucose-containing medium, suggesting that lipase gene expression is prone to catabolite repression. In contrast, lipase production in the lipase-overproducing strain LU8093 was independent of the presence of an inducer and was not inhibited by glucose. In conclusion, hexadecane and Tween 80 enhance lipase production in B. glumae, and they act via different mechanisms.

FIG. 1.

Lipolytic activity in culture supernatant of B. glumae PG1 (A) or LU8093 (B) in the presence of 0% or 10% of hexadecane at different time points. The averages from at least three experiments are shown, with error bars indicating the standard deviations. Asterisks indicate significant differences from sample without hexadecane (P < 0.05).

FIG. 2.

Influence of overexpression of lipase and/or Lif on lipolytic activity. (A) Lipolytic activity in culture supernatants of B. glumae LU8093 containing different plasmids or without plasmid (None). pBBR represents the empty vector pBBR1MCS-2, whereas pLE24 and pLE25 contain the lipB gene alone or the entire lipAB operon, respectively. Cells were grown for 24 h in medium with or without 10% hexadecane. The averages of at least three experiments are shown, with error bars indicating the standard deviations. Asterisks indicate significant difference from the sample without hexadecane (P < 0.05). (B) Western blots of culture supernatants and cell lysates of B. glumae LU8093 containing different plasmids were probed with antisera directed against lipase or Lif.

FIG. 3.

Lipolytic activity (left axis) in culture supernatants (white bars) and relative fluorescence measurements (relative fluorescence units [RFU]) (right axis) of transcriptional (pGPG1-TC and pGLU-TC; gray bars) or translational (pGPG1-TL and pGLU-TL; black bars) lipA::gfp fusions in B. glumae PG1 (A) or LU8093 (B). Strains were grown for 24 h in PG medium supplemented with 1% olive oil (oil), 1% olive oil and 10% hexadecane (oil+HD), 0.5% sucrose (sucrose), or 0.5% sucrose and 0.1% Tween 80 (sucrose+Tw). Averages from at least three experiments are shown, with error bars indicating standard deviations. Asterisks indicate significant difference from sample without hexadecane or Tween 80 (P < 0.05).

FIG. 4.

Effect of hexadecane and chloramphenicol on lipase production in B. glumae LU8093. Cells were washed and incubated for 60 min in PG medium containing 1% olive oil, supplemented with nothing (NA), hexadecane (HD), and/or chloramphenicol (Cm). Samples were taken after 0 or 60 min. (A) Lipolytic activity in culture supernatants (U/ml). (B) Western blot of culture supernatants immunostained with lipase-specific antiserum. (C) Western blot of cell lysates probed with antisera directed against lipase. (D) Western blot of cell lysates probed with antisera directed against Lif.

FIG. 5.

Lipolytic activity in culture supernatants of B. glumae PG1 (A) or LU8093 (B) after 24 h of growth. Cells were grown in the presence of 1% olive oil, 0.5% glucose, or 0.5% sucrose supplemented with nothing (white bars), 10% hexadecane (gray bars), or 0.1% Tween 80 (black bars). Averages from at least three experiments are shown, with error bars indicating standard deviations. Asterisks indicate significant difference from sample without hexadecane or Tween 80 (P < 0.05).