초록
<P><B>Abstract</B></P> <P>Lipases are among the most versatile biocatalysts, and are used in a range of industrially relevant bioconversion reactions. However, the production of LipA in recombinant <I>Bacillus subtilis</I> is still limited, due to unresolved issues surrounding the regulation of the expression and secretion systems. In this study, the gene encoding LipA from <I>B. subtilis</I> 168 was expressed in BNA under the control of the P<SUB>43</SUB> and the P<SUB>AE</SUB> promoter. The extracellular lipase activity of the resulting strains BNACL and BNAAL was 7.8 U ml<SUP>−1</SUP> and 12.6 U ml<SUP>−1</SUP>, respectively. To further enhance the expression of LipA, pHP13L was constructed by inserting the P<SUB>AE</SUB>-<I>lip</I> into the shuttle vector pHP13, which produced an extracellular lipase activity of 180.5 U ml<SUP>−1</SUP> of BNA/pHP13L. The strain BNAY8 described in Supplement data which lacks eight extracellular proteins was constructed and the deletion a few of the much weaker secreting proteins had no significant effect on the secretion of LipA. Moreover, the four Sec pathway components, <I>secA-prfB</I>, <I>secDF</I>, <I>secYEG</I>, <I>prsA</I>, were individually overexpressed in BNA. The overexpression of <I>secDF</I> and <I>prsA</I> enhanced the production of LipA by 28% and 49%, respectively. Furthermore, the co-overexpression of <I>secDF</I> with <I>prsA</I> improved the extracellular amount of LipA by 59% over that of BNA/pHP13L, reaching 287.8 U ml<SUP>−1</SUP>. It can therefore be said that both regulatory elements and secretion pathway had an impact on the production of secreted LipA. Their optimization and modification is a useful strategy to improve the homologous overproduction of other extracellular proteins in <I>B. subtilis</I>.</P> <P><B>Highlights</B></P> <P> <UL> <LI> Using the strong constitutive promoter P<SUB>AE</SUB> without inducer for industrial production of LipA. </LI> <LI> LipA was overexpressed from a plasmid with P<SUB>AE</SUB> reaching 180.5 U ml<SUP>−1</SUP> in the culture supernatant. </LI> <LI> Overexpression of the Sec pathway components <I>secDF</I> and <I>prsA</I> further increased LipA secretion. </LI> <LI> Co-overexpression of <I>secDF</I> and <I>prsA</I> may be a strategy to improve the secretion of other proteins. </LI> </UL> </P>